Methods and compositions for the specific inhibition of gene expression by double-stranded RNA

ABSTRACT

The invention is directed to compositions and methods for selectively reducing the expression of a gene product from a desired target gene in a cell, as well as for treating diseases caused by the expression of the gene. More particularly, the invention is directed to compositions that contain double stranded RNA (“dsRNA”), and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. The dsRNA has a first oligonucleotide sequence that is between 25 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of at least 19 nucleotides is sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to trigger the destruction of the target RNA by the RNAi machinery.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is continuation of U.S. patent application Ser.No. 12/143,002 filed 20 Jun. 2008, which in turn is a division of U.S.patent application Ser. No. 11/797,216 filed 1 May 2007. Each of theseapplications is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

The present invention was made in part with Government support underGrant Numbers AI29329 and HL074704 awarded by the National Institute ofHealth. The Government may have certain rights in this invention.

SEQUENCE LISTING SUBMISSION

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is entitled 1954563SequenceListing, created on 7 Jun. 2012 and is 57 kb in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedbibliography.

The present invention pertains to compositions and methods forgene-specific inhibition of gene expression by double-strandedribonucleic acid (dsRNA) effector molecules. The compositions andmethods are useful in modulating gene expression in a variety ofapplications, including therapeutic, diagnostic, target validation, andgenomic discovery.

BACKGROUND OF THE INVENTION

Suppression of gene expression by double-stranded RNA (dsRNA) has beendemonstrated in a variety of systems including plants(post-transcriptional gene suppression) (Napoli et al., 1990), fungi(quelling) (Romano and Marcino, 1992), and nematodes (RNA interference)(Fire et al., 1998). Double-stranded RNA (dsRNA) is significantly morestable than single-stranded RNA (ssRNA). This difference is pronouncedin the intracellular environment (Raemdonck et al., 2006). However,unmodified siRNAs are rapidly degraded in serum, which is a fairlynuclease rich environment. Chemical modification can significantlystabilize the siRNA and improve potency both in vitro and in vivo.Extensive medicinal chemistry has been done over the past 20 years forapplications where synthetic nucleic acids are used for experimental ortherapeutic applications in vivo, such as in the antisense and ribozymefields, and hundreds of compounds have been tested in a search formodifications that improve nuclease stability, increase bindingaffinity, and sometimes also improve pharmacodynamic properties ofsynthetic nucleic acids (Matteucci, 1997; Manoharan, 2002; Kurreck,2003). Many of these modifications have already been tested and found tohave utility as modifiers for use in traditional 21 mer siRNAs. Severalreviews have provided summaries of recent experience with 21 mer siRNAsand chemical modifications (Zhang et al., 2006; Nawrot and Sipa, 2006;Rana, 2007). Modification patterns have not been tested or optimized foruse in longer RNAs, such as Dicer-substrate siRNAs (DsiRNAs).

Early attempts to suppress gene expression using long dsRNAs inmammalian systems failed due to activation of interferon pathways thatdo not exist in lower organisms. Interferon responses are triggered bydsRNAs (Stark et al., 1998). In particular, the protein kinase PKR isactivated by dsRNAs of greater than 30 bp long (Manche et al., 1992) andresults in phosphorylation of translation initiation factor eIF2α whichleads to arrest of protein synthesis and activation of2′5′-oligoadenylate synthetase (2′-5′-OAS), which leads to RNAdegradation (Minks et al., 1979).

In Drosophila cells and cell extracts, dsRNAs of 150 bp length orgreater were seen to induce RNA interference while shorter dsRNAs wereineffective (Tuschl et al., 1999). Long double-stranded RNA, however, isnot the active effecter molecule; long dsRNAs are degraded by an RNaseIII class enzyme called Dicer (Bernstein et al., 2001) into very short21-23 bp duplexes that have 2-base 3′-overhangs (Zamore et al., 2000).These short RNA duplexes, called siRNAs, direct the RNAi response invivo and transfection of short chemically synthesized siRNA duplexes ofthis design permits use of RNAi methods to suppress gene expression inmammalian cells without triggering unwanted interferon responses(Elbashir et al., 2001a). The antisense strand of the siRNA duplexserves as a sequence-specific guide that directs activity of anendoribonuclease function in the RNA induced silencing complex (RISC) todegrade target mRNA (Martinez et al., 2002).

In studying the size limits for RNAi in Drosophila embryo extracts invitro, a lower threshold of around 38 bp double-stranded RNA wasestablished for activation of RNA interference using exogenouslysupplied double-stranded RNA and duplexes of 36, 30, and 29 bp length(Elbashir et al., 2001b). The short 30-base RNAs were not cleaved intoactive 21-23-base siRNAs and therefore were deemed inactive for use inRNAi (Elbashir et al., 2001b). Continuing to work in the Drosophilaembryo extract system, the same group later carefully mapped thestructural features needed for short chemically synthesized RNA duplexesto function as siRNAs in RNAi pathways. RNA duplexes of 21-bp lengthwith 2-base 3′-overhangs were most effective, duplexes of 20, 22, and23-bp length had slightly decreased potency but did result in RNAimediated mRNA degradation, and 24 and 25-bp duplexes were inactive(Elbashir et al., 2001c).

Some of the conclusions of these earlier studies may be specific to theDrosophila system employed. Other investigators established that longersiRNAs can work in human cells. However, duplexes in the 21-23-bp rangehave been shown to be more active and have become the accepted design(Caplen et al., 2001). Essentially, chemically synthesized duplex RNAsthat mimicked the natural products that result from Dicer degradation oflong duplex RNAs were identified to be the preferred compound for use inRNAi. Approaching this problem from the opposite direction,investigators studying size limits for RNAi in Caenorhabditis elegansfound that although a microinjected 26-bp RNA duplex could function tosuppress gene expression, it required a 250-fold increase inconcentration compared with an 81-bp duplex (Parrish et al., 2000).

Despite the attention given to RNAi research recently, the field isstill in the early stages of development. Not all siRNA molecules arecapable of targeting the destruction of their complementary RNAs in acell. As a result, complex sets of rules have been developed fordesigning RNAi molecules that will be effective. Those having skill inthe art expect to test multiple siRNA molecules to find functionalcompositions. (Ji et al., 2003) Some artisans pool several siRNApreparations together to increase the chance of obtaining silencing in asingle study. (Ji et al., 2003) Such pools typically contain 20 nM of amixture of siRNA oligonucleotide duplexes or more (Ji et al., 2003),despite the fact that a siRNA molecule can work at concentrations of 1nM or less (Holen et al., 2002). This technique can lead to artifactscaused by interactions of the siRNA sequences with other cellular RNAs(“off target effects”). (Scherer et al., 2003) Off target effects canoccur when the RNAi oligonucleotides have homology to unintended targetsor when the RISC complex incorporates the unintended strand from andRNAi duplex. (Scherer et al., 2003) Generally, these effects tend to bemore pronounced when higher concentrations of RNAi duplexes are used.(Scherer et al., 2003)

In addition, the duration of the effect of an effective RNAi treatmentis limited to about 4 days (Holen et al., 2002). Thus, researchers mustcarry out siRNA experiments within 2-3 days of transfection with ansiRNA duplex or work with plasmid or viral expression vectors to obtainlonger term silencing.

One major factor that inhibits the effect of siRNAs is the degradationof siRNAs by nucleases. A 3′-exonuclease is the primary nucleaseactivity present in serum and modification of the 3′-ends of antisenseDNA oligonucleotides is crucial to prevent degradation (Eder et al.,1991). An RNase-T family nuclease has been identified called ERI-1 whichhas 3′→5′ exonuclease activity that is involved in regulation anddegradation of siRNAs (Kennedy et al., 2004; Hong et al., 2005). Thisgene is also known as Thex1 (NM_(—)02067) in mice or THEX1(NM_(—)153332) in humans and is involved in degradation of histone mRNA;it also mediates degradation of 3′-overhangs in siRNAs, but does notdegrade duplex RNA (Yang et al., 2006). It is therefore reasonable toexpect that 3′-end-stabilization of siRNAs will improve stability.

XRN1 (NM_(—)019001) is a 5′→3′ exonuclease that resides in P-bodies andhas been implicated in degradation of mRNA targeted by miRNA (Rehwinkelet al., 2005) and may also be responsible for completing degradationinitiated by internal cleavage as directed by a siRNA. XRN2(NM_(—)012255) is a distinct 5′→3′ exonuclease that is involved innuclear RNA processing. Although not currently implicated in degradationor processing of siRNAs and miRNAs, these both are known nucleases thatcan degrade RNAs and may also be important to consider.

RNase A is a major endonuclease activity in mammals that degrades RNAs.It is specific for ssRNA and cleaves at the 3′-end of pyrimidine bases.SiRNA degradation products consistent with RNase A cleavage can bedetected by mass spectrometry after incubation in serum (Turner et al.,2007). The 3′-overhangs enhance the susceptibility of siRNAs to RNasedegradation. Depletion of RNase A from serum reduces degradation ofsiRNAs; this degradation does show some sequence preference and is worsefor sequences having poly A/U sequence on the ends (Haupenthal et al.,2006). This suggests the possibility that lower stability regions of theduplex may “breathe” and offer transient single-stranded speciesavailable for degradation by RNase A. RNase A inhibitors can be added toserum and improve siRNA longevity and potency (Haupenthal et al., 2007).

In 21 mers, phosphorothioate or boranophosphate modifications directlystabilize the internucleoside phosphate linkage. Boranophosphatemodified RNAs are highly nuclease resistant, potent as silencing agents,and are relatively non-toxic. Boranophosphate modified RNAs cannot bemanufactured using standard chemical synthesis methods and instead aremade by in vitro transcription (IVT) (Hall et al., 2004 and Hall et al.,2006). Phosphorothioate (PS) modifications can be easily placed in theRNA duplex at any desired position and can be made using standardchemical synthesis methods. The PS modification shows dose-dependenttoxicity, so most investigators have recommended limited incorporationin siRNAs, favoring the 3′-ends where protection from nucleases is mostimportant (Harborth et al., 2003; Chiu and Rana, 2003; Braasch et al.,2003; Amarzguioui et al., 2003). More extensive PS modification can becompatible with potent RNAi activity; however, use of sugarmodifications (such as 2′-O-methyl RNA) may be superior (Choung et al.,2006).

A variety of substitutions can be placed at the 2′-position of theribose which generally increases duplex stability (T_(m)) and cangreatly improve nuclease resistance. 2′-O-methyl RNA is a naturallyoccurring modification found in mammalian ribosomal RNAs and transferRNAs. 2′-O-methyl modification in siRNAs is known, but the preciseposition of modified bases within the duplex is important to retainpotency and complete substitution of 2′-O-methyl RNA for RNA willinactivate the siRNA. For example, a pattern that employs alternating2′-O-methyl bases can have potency equivalent to unmodified RNA and isquite stable in serum (Choung et al., 2006; Czauderna et al., 2003).

The 2′-fluoro (2′-F) modification is also compatible with siRNAfunction; it is most commonly placed at pyrimidine sites (due to reagentcost and availability) and can be combined with 2′-O-methyl modificationat purine positions; 2′-F purines are available and can also be used.Heavily modified duplexes of this kind can be potent triggers of RNAi invitro (Allerson et al., 2005; Prakash et al., 2005; Kraynack and Baker,2006) and can improve performance and extend duration of action whenused in vivo (Morrissey et al., 2005a; Morrissey et al., 2005b). Ahighly potent, nuclease stable, blunt 19 mer duplex containingalternative 2′-F and 2′-O-Me bases is taught by Allerson. In thisdesign, alternating 2′-O-Me residues are positioned in an identicalpattern to that employed by Czauderna, however the remaining RNAresidues are converted to 2′-F modified bases. A highly potent, nucleaseresistant siRNA employed by Morrissey employed a highly potent, nucleaseresistant siRNA in vivo. In addition to 2′-O-Me RNA and 2′-F RNA, thisduplex includes DNA, RNA, inverted abasic residues, and a 3′-terminal PSinternucleoside linkage. While extensive modification has certainbenefits, more limited modification of the duplex can also improve invivo performance and is both simpler and less costly to manufacture.Soutschek et al. (2004) employed a duplex in vivo and was mostly RNAwith two 2′-O-Me RNA bases and limited 3′-terminal PS internucleosidelinkages.

Locked nucleic acids (LNAs) are a different class of 2′-modificationthat can be used to stabilize siRNAs. Patterns of LNA incorporation thatretain potency are more restricted than 2′-O-methyl or 2′-F bases, solimited modification is preferred (Braasch et al., 2003; Grunweller etal., 2003; Elmen et al., 2005). Even with limited incorporation, the useof LNA modifications can improve siRNA performance in vivo and may alsoalter or improve off target effect profiles (Mook et al., 2007).

Synthetic nucleic acids introduced into cells or live animals can berecognized as “foreign” and trigger an immune response. Immunestimulation constitutes a major class of off-target effects which candramatically change experimental results and even lead to cell death.The innate immune system includes a collection of receptor moleculesthat specifically interact with DNA and RNA that mediate theseresponses, some of which are located in the cytoplasm and some of whichreside in endosomes (Marques and Williams, 2005; Schlee et al., 2006).Delivery of siRNAs by cationic lipids or liposomes exposes the siRNA toboth cytoplasmic and endosomal compartments, maximizing the risk fortriggering a type 1 interferon (IFN) response both in vitro and in vivo(Morrissey et al., 2005b; Sioud and Sorensen, 2003; Sioud, 2005; Ma etal., 2005). RNAs transcribed within the cell are less immunogenic(Robbins et al., 2006) and synthetic RNAs that are immunogenic whendelivered using lipid-based methods can evade immune stimulation whenintroduced unto cells by mechanical means, even in vivo (Heidel et al.,2004). However, lipid based delivery methods are convenient, effective,and widely used. Some general strategy to prevent immune responses isneeded, especially for in vivo application where all cell types arepresent and the risk of generating an immune response is highest. Use ofchemically modified RNAs may solve most or even all of these problems.

Although certain sequence motifs are clearly more immunogenic thanothers, it appears that the receptors of the innate immune system ingeneral distinguish the presence or absence of certain basemodifications which are more commonly found in mammalian RNAs than inprokaryotic RNAs. For example, pseudouridine, N6-methyl-A, and2′-O-methyl modified bases are recognized as “self” and inclusion ofthese residues in a synthetic RNA can help evade immune detection(Kariko et al., 2005). Extensive 2′-modification of a sequence that isstrongly immunostimulatory as unmodified RNA can block an immuneresponse when administered to mice intravenously (Morrissey et al.,2005b). However, extensive modification is not needed to escape immunedetection and substitution of as few as two 2′-O-methyl bases in asingle strand of a siRNA duplex can be sufficient to block a type 1 IFNresponse both in vitro and in vivo; modified U and G bases are mosteffective (Judge et al., 2006). As an added benefit, selectiveincorporation of 2′-O-methyl bases can reduce the magnitude ofoff-target effects (Jackson et al., 2006). Use of 2′-O-methyl basesshould therefore be considered for all siRNAs intended for in vivoapplications as a means of blocking immune responses and has the addedbenefit of improving nuclease stability and reducing the likelihood ofoff-target effects.

Although cell death can results from immune stimulation, assessing cellviability is not an adequate method to monitor induction of IFNresponses. IFN responses can be present without cell death, and celldeath can result from target knockdown in the absence of IFN triggering(for example, if the targeted gene is essential for cell viability).Relevant cytokines can be directly measured in culture medium and avariety of commercial kits exist which make performing such assaysroutine. While a large number of different immune effector molecules canbe measured, testing levels of IFN-α, TNF-α, and IL-6 at 4 and 24 hourspost transfection is usually sufficient for screening purposes. It isimportant to include a “transfection reagent only control” as cationiclipids can trigger immune responses in certain cells in the absence ofany nucleic acid cargo. Including controls for IFN pathway inductionshould be considered for cell culture work. It is essential to test forimmune stimulation whenever administering nucleic acids in vivo, wherethe risk of triggering IFN responses is highest.

There is therefore a need to provide a chemical modification patternthat would enhance the efficacy of a dsRNA, particularly DsiRNA. Themodifications 1) should not reduce potency; 2) should not interfere withDicer processing; 3) should improve stability in biological fluids(reduce nuclease sensitivity); 4) should block or evade detection by theinnate immune system; 5) should not be toxic; and 6) should not increasecost or impact ease of manufacturing.

The invention provides compositions useful in RNAi for inhibiting geneexpression and provides methods for their use. In addition, theinvention provides RNAi compositions and methods designed to maximizepotency, enhance Dicer processing, improve stability while evading theimmune system and are not toxic. Additionally, various embodiments ofthe invention are suited for high throughput, small scale synthesis tomeet research needs as well as large scale manufacturing for therapeuticapplications. These and other advantages of the invention, as well asadditional inventive features, will be apparent from the description ofthe invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to compositions that contain doublestranded RNA (“dsRNA”), and methods for preparing them, that are capableof reducing the expression of target genes in eukaryotic cells. Moreparticularly, the invention is directed to Dicer substrate RNAs withmodifications that are functionally improved.

Thus, in a first aspect, the present invention provides novelcompositions for RNA interference (RNAi). The compositions comprisedsRNA which is a precursor molecule, i.e., the dsRNA of the presentinvention is processed in vivo to produce an active siRNA. The dsRNA isprocessed by Dicer to an active siRNA which is incorporated into theRISC complex for RNA interference of a target gene. The precursormolecule is also termed a precursor RNAi molecule herein.

In one embodiment, the dsRNA, i.e., the precursor RNAi molecule, has alength sufficient such that it is processed by Dicer to produce ansiRNA. According to this embodiment, the dsRNA comprises a firstoligonucleotide sequence (also termed the sense strand) that is between26 and about 30 nucleotides in length and a second oligonucleotidesequence (also termed the antisense strand) that anneals to the firstsequence under biological conditions, such as the conditions found inthe cytoplasm of a cell. In addition, a region of one of the sequences,particularly of the antisense strand, of the dsRNA has a sequence lengthof at least 19 nucleotides, for example, from about 19 to about 23nucleotides, such as 21 nucleotides that are sufficiently complementaryto a nucleotide sequence of the RNA produced from the target gene totrigger an RNAi response.

In a second embodiment, the dsRNA, i.e., the precursor RNAi molecule,has several properties which enhance its processing by Dicer. Accordingto this embodiment, the dsRNA has a length sufficient such that it isprocessed by Dicer to produce an siRNA and at least one of the followingproperties: (i) the dsRNA is asymmetric, e.g., has a 3′ overhang on theantisense strand and (ii) the dsRNA has a modified 3′ end on the sensestrand to direct orientation of Dicer binding and processing of thedsRNA to an active siRNA. According to this embodiment, the sense strandcomprises 22-28 nucleotides and the antisense strand comprises 24-30nucleotides. In one embodiment, the dsRNA has an overhang on the 3′ endof the antisense strand. In another embodiment, the sense strand ismodified for Dicer binding and processing by suitable modifiers locatedat the 3′ end of the sense strand. Suitable modifiers includenucleotides such as deoxyribonucleotides, acyclonucleotides and the likeand sterically hindered molecules, such as fluorescent molecules and thelike. When nucleotide modifiers are used, they replace ribonucleotidesin the dsRNA such that the length of the dsRNA does not change. Inanother embodiment, the dsRNA has an overhang on the 3′ end of theantisense strand and the sense strand is modified for Dicer processing.In another embodiment, the 5′ end of the sense strand has a phosphate.In another embodiment, the 5′ end of the antisense strand has aphosphate. In another embodiment, the antisense strand or the sensestrand or both strands have one or more 2′-O-methyl modifiednucleotides. In another embodiment, the antisense strand contains2′-O-methyl modified nucleotides. In another embodiment, the antisensestand contains a 3′ overhang that is comprised of 2′-O-methyl modifiednucleotides. The antisense strand could also include additional2′-O-methyl modified nucleotides. The sense and antisense strands annealunder biological conditions, such as the conditions found in thecytoplasm of a cell. In addition, a region of one of the sequences,particularly of the antisense strand, of the dsRNA has a sequence lengthof at least 19 nucleotides, wherein these nucleotides are in the21-nucleotide region adjacent to the 3′ end of the antisense strand andare sufficiently complementary to a nucleotide sequence of the RNAproduced from the target gene. Further in accordance with thisembodiment, the dsRNA, i.e., the precursor RNAi molecule, may also haveone or more of the following additional properties: (a) the antisensestrand has a right shift from the typical 21 mer (i.e., the antisensestrand includes nucleotides on the right side of the molecule whencompared to the typical 21 mer), (b) the strands may not be completelycomplementary, i.e., the strands may contain simple mismatch pairingsand (c) base modifications such as locked nucleic acid(s) may beincluded in the 5′ end of the sense strand.

In a third embodiment, the sense strand comprises 25-28 nucleotides,wherein the 2 nucleotides on the 3′ end of the sense strand aredeoxyribonucleotides. The sense strand contains a phosphate at the 5′end. The antisense strand comprises 26-30 nucleotides and contains a 3′overhang of 1-4 nucleotides. The nucleotides comprising the 3′ overhangare modified with 2′-O-methyl RNA. The antisense strand containsalternating 2′-O-methyl modified nucleotides beginning at the firstmonomer of the antisense strand adjacent to the 3′ overhang, andextending 15-19 nucleotides from the first monomer adjacent to the 3′overhang. For example, for a 27 nucleotide antisense strand and countingthe first base at the 5′-end of the antisense strand as position number1, 2′OMe modifications would be placed at bases 9, 11, 13, 15, 17, 19,21, 23, 25, 26, and 27. In one embodiment, the DsiRNA comprises:

5′ pXXXXXXXXXXXXXXXXXXXXXXXDD 3′ Y X X X X X X X X X X X X X X X X XXXXXXXXXpwherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sensestrand, and the bottom strand is the antisense strand.

In a fourth embodiment, the dsRNA, i.e., the precursor RNAi molecule,has several properties which enhance its processing by Dicer. Accordingto this embodiment, the dsRNA has a length sufficient such that it isprocessed by Dicer to produce an siRNA and at least one of the followingproperties: (i) the dsRNA is asymmetric, e.g., has a 3′ overhang on thesense strand and (ii) the dsRNA has a modified 3′ end on the antisensestrand to direct orientation of Dicer binding and processing of thedsRNA to an active siRNA. According to this embodiment, the sense strandcomprises 24-30 nucleotides and the antisense strand comprises 22-28nucleotides. In one embodiment, the dsRNA has an overhang on the 3′ endof the sense strand. In another embodiment, the antisense strand ismodified for Dicer binding and processing by suitable modifiers locatedat the 3′ end of the antisense strand. Suitable modifiers includenucleotides such as deoxyribonucleotides, acyclonucleotides and the likeand sterically hindered molecules, such as fluorescent molecules and thelike. When nucleotide modifiers are used, they replace ribonucleotidesin the dsRNA such that the length of the dsRNA does not change. Inanother embodiment, the dsRNA has an overhang on the 3′ end of the sensestrand and the antisense strand is modified for Dicer processing. In oneembodiment, the antisense strand has a 5′ phosphate. The sense andantisense strands anneal under biological conditions, such as theconditions found in the cytoplasm of a cell. In addition, a region ofone of the sequences, particularly of the antisense strand, of the dsRNAhas a sequence length of at least 19 nucleotides, wherein thesenucleotides are adjacent to the 3′ end of antisense strand and aresufficiently complementary to a nucleotide sequence of the RNA producedfrom the target gene. Further in accordance with this embodiment, thedsRNA, i.e., the precursor RNAi molecule, may also have one or more ofthe following additional properties: (a) the antisense strand has a leftshift from the typical 21 mer (i.e., the antisense strand includesnucleotides on the left side of the molecule when compared to thetypical 21 mer) and (b) the strands may not be completely complementary,i.e., the strands may contain simple mismatch pairings.

In a second aspect, the present invention provides a method for making adsRNA, i.e., a precursor RNAi molecule, that has enhanced processing byDicer. According to this method an antisense strand siRNA having alength of at least 19 nucleotides is selected for a given target geneusing conventional techniques and algorithms. In one embodiment, theantisense siRNA is modified to include 5-11 ribonucleotides on the 5′end to give a length of 24-30 nucleotides. When the antisense strand hasa length of 21 nucleotides, then 3-9 nucleotides, or 4-7 nucleotides or6 nucleotides are added on the 5′ end. Although the addedribonucleotides may be complementary to the target gene sequence, fullcomplementarity between the target sequence and the antisense siRNA isnot required. That is, the resultant antisense siRNA is sufficientlycomplementary with the target sequence. A sense strand is then producedthat has 22-28 nucleotides. The sense strand is substantiallycomplementary with the antisense strand to anneal to the antisensestrand under biological conditions. In one embodiment, the sense strandis synthesized to contain a modified 3′ end to direct Dicer processingof the antisense strand. In another embodiment, the antisense strand ofthe dsRNA has a 3′ overhang. In a further embodiment, the sense strandis synthesized to contain a modified 3′ end for Dicer binding andprocessing and the antisense strand of the dsRNA has a 3′ overhang.

In a second embodiment of this method, the antisense siRNA is modifiedto include 1-9 ribonucleotides on the 5′ end to give a length of 22-28nucleotides. When the antisense strand has a length of 21 nucleotides,then 1-7 ribonucleotides, or 2-5 ribonucleotides and or 4ribonucleotides are added on the 3′ end. The added ribonucleotides mayhave any sequence. Although the added ribonucleotides may becomplementary to the target gene sequence, full complementarity betweenthe target sequence and the antisense siRNA is not required. That is,the resultant antisense siRNA is sufficiently complementary with thetarget sequence. A sense strand is then produced that has 24-30nucleotides. The sense strand is substantially complementary with theantisense strand to anneal to the antisense strand under biologicalconditions. In one embodiment, the antisense strand is synthesized tocontain a modified 3′ end to direct Dicer processing. In anotherembodiment, the sense strand of the dsRNA has a 3′ overhang. In afurther embodiment, the antisense strand is synthesized to contain amodified 3′ end for Dicer binding and processing and the sense strand ofthe dsRNA has a 3′ overhang.

In a third aspect, the present invention provides pharmaceuticalcompositions containing the disclosed dsRNA compositions.

In a fourth aspect, the present invention provides methods forselectively reducing the expression of a gene product from a desiredtarget gene in a cell, as well as for treating diseases caused by theexpression of the gene. In one embodiment, the method involvesintroducing into the environment of a cell an amount of a dsRNA of thepresent invention such that a sufficient portion of the dsRNA can enterthe cytoplasm of the cell to cause a reduction in the expression of thetarget gene.

The compositions and methods have an unanticipated level of potency ofthe RNAi effect. Although the invention is not intended to be limited bythe underlying theory on which it is believed to operate, it is thoughtthat this level of potency and duration of action are caused by the factthe dsRNA serves as a substrate for Dicer which appears to facilitateincorporation of one sequence from the dsRNA into the RISC complex thatis directly responsible for destruction of the RNA from the target gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show that 27 mer dsRNAs are more potent effectors of RNAithan a 21+2 siRNA. EGFP expression levels were determined aftercotransfection of HEK293 cells with a fixed amount of EGFP expressionplasmid and varying concentrations of dsRNAs. FIGS. 1A-1C: Transfectionswere performed using (FIG. 1A) 50 nM, (FIG. 1B) 200 pM and (FIG. 1C) 50pM of the indicated dsRNAs. FIG. 1D: Dose-response testing of longerdsRNAs. Transfections were performed with the indicated concentrationsof dsRNA. FIG. 1E: Depicts in vitro Dicer reactions with the same longerRNAs. Concentrations and conditions were as described in the Examples.FIG. 1F: Dose-response curve of dsRNAs transfected into NIH3T3 cellsthat stably express EGFP. Each graph point represents the average (withs.d.) of three independent measurements.

FIGS. 2A-2E show that Dicer processing correlates with RNAi activity.FIG. 2A: Cleavage of dsRNAs by recombinant Dicer. Each RNA duplex wasincubated in the presence or absence of recombinant human Dicer for 24h, separated using nondenaturing PAGE and visualized (see Examples).FIG. 2B: RNA duplexes used in this test. Oligos were conjugated with6FAM at the 5′ ends, the 3′ ends or both as shown by the circles. Topand bottom lines indicate sense and antisense strands in duplexconfiguration, with the sense in a 5′-to-3′ orientation (left to right)and the antisense in a 3′-to-5′ orientation (left to right). FIG. 2C:6FAM end-modification affects in vitro Dicer activity. RNA duplexes wereincubated with 0.5 units of recombinant human Dicer for 8 h and theproducts resolved on a 7.5% nondenaturing polyacrylamide gel. The RNAswere visualized by ethidium bromide staining. FIG. 2D: 6FAM modificationaffects RNAi activity. RNA duplexes at 200 pM were cotransfected withthe EGFP expression plasmid and assayed at 24 h for EGFP fluorescence asdescribed. Reported values for EGFP expression represent the average oftwo independent experiments. The relative levels of fluorescence werenormalized to those for luciferase. FIG. 2E: 27 mer duplex RNAs areprocessed to 21 mers in vivo. Total RNA was prepared from cellstransfected with duplex 3 and duplex 5 at 10 nM. RNA was hybridized witha 21 mer ³²P-labeled oligonucleotide probe. The hybridized samples wereseparated by nondenaturing PAGE and visualized by autoradiography. Sizemarkers are ³²P end labeled 21 mer and 27 mer RNA duplexes.

FIG. 3A-3B show RNAi activity of various 21+2 siRNAs. FIG. 3A: Sevenpossible 21+2 siRNAs predicted from Dicing the 27 mer dsRNA were testedindividually or as a pool in co-transfection assays with the EGFPreporter construct in HEK293 cells. Each graph depicts the average ofduplicate experiments. FIG. 3B: Comparison of in vitro Diced 27 merdsRNA versus intact 27 mer dsRNA for RNAi. The respective RNAs wereco-transfected as in FIG. 4A at the indicated concentrations of dsRNAs.For the Diced products, a 1 μM 27 mer dsRNA was incubated in Dicerreaction buffer without (column 3) or with Dicer (column 4) at 37° C.for 12 hours. The mixtures were diluted in water and used directly forco-transfection with the EGFP reporter. To control for possibleartifacts of residual Dicer in the diluted mixes, the samples in column4 were phenol extracted and ethanol precipitated prior to transfection(column 5).

FIGS. 4A and 4B show ESI mass spectra of the 27 mer duplex EGFPS1 27+0before (FIG. 4A) and after (FIG. 4B) incubation with Dicer. Duplexesseparate into single strands and the measured mass of each strand isindicated. Dicer digestion is performed in the presence of high salt andsome “shadow” peaks represent +1 or +2 Na species.

FIGS. 5A-5D show features of 27 mer dsRNA in RNAi. FIG. 5A: Enhancedduration of RNAi by 27 mer dsRNAs. Levels of EGFP were determined aftertransfection of 5 nM of a 21+2 siRNA or the 27 mer dsRNA into NIH3T3cells stably expressing EGFP. Graphic representation of EGFP silencingmediated by a 21+2 siRNA as compared to the 27 mer dsRNA. Duplicatesamples were taken on the indicated days and EGFP expression wasdetermined by fluorometry. FIG. 5B: 27 mer dsRNAs, targeting sitesrefractory to 21 mer siRNAs, can elicit RNAi. The dsRNAs weretransfected along with the EGFP reporter construct, and EGFP expressionwas determined (Methods). Column 1, mock; column 2, 21+2 siRNA targetingEGFPS2; column 3, 27 mer dsRNA targeting EGFPS2; column 4, 21+2 siRNAtargeting EGFPS3; column 5, 27 mer dsRNA targeting EGFPS3. FIGS. 5C and5D: Comparison of 21 mer siRNA and 27 mer dsRNA in downregulation ofendogenous transcripts. RNAs for a 21+2 siRNA and 27+0 dsRNA weredesigned to target sites in the hnRNP H mRNA (FIG. 5C) or La mRNA (FIG.5D). HnRNP H knockdown was assayed by western blot and La knockdown bynorthern blot analyses. The dsRNAs were used at the indicatedconcentrations. β-Actin was used as an internal specificity and loadingstandard in both experiments.

FIG. 6 shows sequence specificity of Dicer substrate 27 mer dsRNAs. Thevarious 27 mer dsRNAs were co-transfected at the indicatedconcentrations with the EGFP expression plasmid into HEK93 cells andassayed for EGFP fluorescence.

FIGS. 7A-7D show that siRNAs and Dicer substrate dsRNAs do not induceinterferons or activate PKR or generate specific “off target effects.”FIGS. 7A and 7B: Interferon alpha (FIG. 7A) and interferon beta (FIG.7B) assays: column 1, positive control for IFN induction (Kim et al.,2004); column 2, no RNA; column 3, chemically synthesized 21+2 siRNA;column 4, chemically synthesized 27+0 dsRNA. FIG. 7C: PKR activationassay. The lond dsRNA used for PKR activation (Kim et al., 2004) and thein vitro PKR activation assay (Manche et al, 1992) have been previouslydescribed. Duplex RNAs were transfected as indicated. FIG. 7D: Summaryof microarray analysis.

FIGS. 8A-8B show ESI mass spectra of the 27 mer duplex EGFPS1 27+0 Lbefore (FIG. 8A) and after (FIG. 8B) incubation with Dicer. Duplexesseparate into single strands and the measured mass of each strand isindicated.

FIGS. 8C-8D show ESI mass spectra of the 27 mer duplex EGFPS1 27/25 Lbefore (FIG. 8C) and after (FIG. 8D) incubation with Dicer. Duplexesseparate into single strands and the measured mass of each strand isindicated.

FIGS. 9A-9B show ESI mass spectra of the 27 mer duplex EGFPS1 27+0 Rbefore (FIG. 9A) and after (FIG. 9B) incubation with Dicer. Duplexesseparate into single strands and the measured mass of each strand isindicated.

FIGS. 9C-9D show ESI mass spectra of the 27 mer duplex EGFPS1 25/27 Rbefore (FIG. 9C) and after (FIG. 9B) incubation with Dicer. Duplexesseparate into single strands and the measured mass of each strand isindicated.

FIGS. 10A-10B show that duplexes designed to enhance Dicer processingare potent effectors of RNAi. EGFP expression levels were determinedafter cotransfection of HEK293 cells with a fixed amount of EGFPexpression plasmid and varying concentrations of dsRNAs. FIG. 10A:Compares the potency of the duplexes EGFPS2-21+2, EGFPS2-27+0,EGFPS2-27/25 L and EGFPS2-25/27 R. FIG. 10B: Compares the potency of theduplexes EGFPS1-27/25 L and EGFPS1-25/27 R.

FIG. 11 is an illustration showing two embodiments of the presentinvention with respect to the target sequence and the relationshipbetween the target sequence and each embodiment.

FIG. 12 shows the effect that various modifications incorporated into a21 mer duplex can have to the potency of that duplex as a trigger ofRNAi. The figure lists the % mRNA levels relative to an unmodifiedcontrol duplex as assessed by qRT-PCR 24 hours after transfection intoHeLa cells a 1 nM concentration.

FIG. 13 shows the precise site of cleavage within a 25/27 mer dsRNAsubstrate where Dicer processing is expected to occur. The Figure showsthat modifications to the right of the expected cleavage site will notbe present in the final product after cleavage.

FIG. 14 shows the effect that various modifications incorporated into a25/27 mer DsiRNA duplex can have to the potency of that duplex as atrigger of RNAi. The figure lists the % mRNA levels relative to ancontrol duplex modified only with 2 DNA bases on the 3′ end of the sensestrand as assessed by qRT-PCR 24 hours after transfection into HeLacells a 1 nM concentration.

FIG. 15 demonstrates the relative performance of several modificationpatterns on DsiRNAs targeting the EGFP gene sequence. The resultsindicate that all single-strand modified variants worked well inreducing EGFP expression.

FIG. 16 shows the relative potency of several DsiRNA modificationpatterns against the human HPRT gene. Three doses were tested (10 nM, 1nM, and 0.1 nM). The figure demonstrates not only that modified DsiRNAswork as well or nearly as well to unmodified DsiRNAs, but also that25/27 mers can potentially be loaded into RISC without Dicer cleavage.

FIG. 17A shows the relative change in expression levels of a variety ofimmune pathway genes in T98G cells before and after transfection withunmodified vs. ASm modified DsiRNAs. The 2′-O-methyl modified DsiRNAsresulted in minimal alterations in gene expression levels whileunmodified DsiRNAs resulted in over a 200-fold increase in expression ofcertain genes. Relative mRNA levels were assessed by qRT-PCR at 24 hpost transfection. FIGS. 17B and 17C provide finer detail views of thecontrol gene assay results. In 17B, unmodified HPRT-specific DsiRNAsresulted in reduction of HPRT mRNA levels as well as a reduction of themRNA levels of a control non-targeted housekeeping gene RPLP0, which ischaracteristic of a Type-I IFN response. In 17C, the ASm modifiedHPRT-specific DsiRNA resulted in reduction of HPRT mRNA levels with nochange in expression of the control RPLP0 gene.

FIG. 18 demonstrates the change in secreted cytokine levels when 25/27mer DsiRNAs are introduced into T98G cells in tissue culture.Supernatants from the same cell cultures reported in Example 20, FIG. 17for gene expression levels were assayed for the levels of variouscytokines. Transfection of an unmodified HPRT DsiRNA resulted insignificant elevations of IL-8 and IL-6 cytokine levels, indicative ofstrong stimulation of an immune signaling pathway. Transfection of thesame sequence modified with 2′-O-methyl RNA in the ASm pattern did notshow significant elevation of any cytokines.

FIG. 19 demonstrates the improved stability of modified 27 mer DsiRNAdesigns compared to unmodified DsiRNA and 21 mer siRNA duplexes. RNAduplexes were incubated at 37° C. for the indicated length of time in50% fetal bovine serum, extracted, and separated on polyacrylamide gelelectrophoresis. Unmodified 27 mer DsiRNA duplexes showed improvedstability compared with unmodified 21 mer duplexes and best stabilitywas seen with the 2′-O-methyl ASm+5′P modification pattern.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions that contain doublestranded RNA (“dsRNA”), and methods for preparing them, that are capableof reducing the expression of target genes in eukaryotic cells. One ofthe strands of the dsRNA contains a region of nucleotide sequence thathas a length that ranges from about 19 to about 30 nucleotides that candirect the destruction of the RNA transcribed from the target gene.

In a first aspect, the present invention provides novel compositions forRNA interference (RNAi). The compositions comprise dsRNA which is aprecursor molecule, i.e., the dsRNA of the present invention isprocessed in vivo to produce an active siRNA. The dsRNA is processed byDicer to an active siRNA which is incorporated into the RISC complex.The precursor molecule is also termed a precursor RNAi molecule herein.As used herein, the term active siRNA refers to a double strandednucleic acid in which each strand comprises RNA, RNA analog(s) or RNAand DNA. The siRNA comprises between 19 and 23 nucleotides or comprises21 nucleotides. The active siRNA has 2 bp overhangs on the 3′ ends ofeach strand such that the duplex region in the siRNA comprises 17-21nucleotides, or 19 nucleotides. Typically, the antisense strand of thesiRNA is sufficiently complementary with the target sequence of thetarget gene.

The phrase “duplex region” refers to the region in two complementary orsubstantially complementary oligonucleotides that form base pairs withone another, either by Watson-Crick base pairing or any other mannerthat allows for a duplex between oligonucleotide strands that arecomplementary or substantially complementary. For example, anoligonucleotide strand having 21 nucleotide units can base pair withanother oligonucleotide of 21 nucleotide units, yet only 19 bases oneach strand are complementary or substantially complementary, such thatthe “duplex region” consists of 19 base pairs. The remaining base pairsmay, for example, exist as 5′ and 3′ overhangs. Further, within theduplex region, 100% complementarity is not required; substantialcomplementarity is allowable within a duplex region. Substantialcomplementarity refers to complementarity between the strands such thatthey are capable of annealing under biological conditions. Techniques toempirically determine if two strands are capable of annealing underbiological conditions are well know in the art. Alternatively, twostrands can be synthesized and added together under biologicalconditions to determine if they anneal to one another.

As used herein, a siRNA having a sequence “sufficiently complementary”to a target mRNA sequence means that the siRNA has a sequence sufficientto trigger the destruction of the target mRNA by the RNAi machinery(e.g., the RISC complex) or process. The siRNA molecule can be designedsuch that every residue of the antisense strand is complementary to aresidue in the target molecule. Alternatively, substitutions can be madewithin the molecule to increase stability and/or enhance processingactivity of said molecule. Substitutions can be made within the strandor can be made to residues at the ends of the strand.

In one embodiment of the first aspect of the present invention, thedsRNA, i.e., the precursor RNAi molecule, has a length sufficient suchthat it is processed by Dicer to produce an siRNA. According to thisembodiment, a suitable dsRNA contains one oligonucleotide sequence, afirst sequence, that is at least 25 nucleotides in length and no longerthan about 30 nucleotides. This sequence of RNA can be between about 26and 29 nucleotides in length. This sequence can be about 27 or 28nucleotides in length or 27 nucleotides in length. The second sequenceof the dsRNA can be any sequence that anneals to the first sequenceunder biological conditions, such as within the cytoplasm of aeukaryotic cell. Generally, the second oligonucleotide sequence willhave at least 19 complementary base pairs with the first oligonucleotidesequence, more typically the second oligonucleotides sequence will haveabout 21 or more complementary base pairs, or about 25 or morecomplementary base pairs with the first oligonucleotide sequence. In oneembodiment, the second sequence is the same length as the firstsequence, and the dsRNA is blunt ended. In another embodiment, the endsof the dsRNA have overhangs.

In certain aspects of this first embodiment, the first and secondoligonucleotide sequences of the dsRNA exist on separate oligonucleotidestrands that can be and typically are chemically synthesized. In someembodiments, both strands are between 26 and 30 nucleotides in length.In other embodiments, both strands are between 25 and 30 nucleotides inlength. In one embodiment, both strands are 27 nucleotides in length,are completely complementary and have blunt ends. The dsRNA can be froma single RNA oligonucleotide that undergoes intramolecular annealing or,more typically, the first and second sequences exist on separate RNAoligonucleotides. In one embodiment, one or both oligonucleotide strandsare capable of serving as a substrate for Dicer. In other embodiments,at least one modification is present that promotes Dicer to bind to thedouble-stranded RNA structure in an orientation that maximizes thedouble-stranded RNA structure's effectiveness in inhibiting geneexpression. The dsRNA can contain one or more deoxyribonucleic acid(DNA) base substitutions.

Suitable dsRNA compositions that contain two separate oligonucleotidescan be chemically linked outside their annealing region by chemicallinking groups. Many suitable chemical linking groups are known in theart and can be used. Suitable groups will not block Dicer activity onthe dsRNA and will not interfere with the directed destruction of theRNA transcribed from the target gene. Alternatively, the two separateoligonucleotides can be linked by a third oligonucleotide such that ahairpin structure is produced upon annealing of the two oligonucleotidesmaking up the dsRNA composition. The hairpin structure will not blockDicer activity on the dsRNA and will not interfere with the directeddestruction of the RNA transcribed from the target gene.

The first and second oligonucleotides are not required to be completelycomplementary. In fact, in one embodiment, the 3′-terminus of the sensestrand contains one or more mismatches. In one aspect, about twomismatches are incorporated at the 3′ terminus of the sense strand. Inanother embodiment, the dsRNA of the invention is a double stranded RNAmolecule containing two RNA oligonucleotides each of which is 27nucleotides in length and, when annealed to each other, have blunt endsand a two nucleotide mismatch on the 3′-terminus of the sense strand(the 5′-terminus of the antisense strand). The use of mismatches ordecreased thermodynamic stability (specifically at the3′-sense/5′-antisense position) has been proposed to facilitate or favorentry of the antisense strand into RISC (Schwarz et al., 2003; Khvorovaet al., 2003), presumably by affecting some rate-limiting unwindingsteps that occur with entry of the siRNA into RISC. Thus, terminal basecomposition has been included in design algorithms for selecting active21 mer siRNA duplexes (Ui-Tei et al., 2004; Reynolds et al., 2004). WithDicer cleavage of the dsRNA of this embodiment, the small end-terminalsequence which contains the mismatches will either be left unpaired withthe antisense strand (become part of a 3′-overhang) or be cleavedentirely off the final 21-mer siRNA. These “mismatches”, therefore, donot persist as mismatches in the final RNA component of RISC. It wassurprising to find that base mismatches or destabilization of segmentsat the 3′-end of the sense strand of Dicer substrate improved thepotency of synthetic duplexes in RNAi, presumably by facilitatingprocessing by Dicer.

It has been found empirically that these longer dsRNA species of from 25to about 30 nucleotides give unexpectedly effective results in terms ofpotency and duration of action. Without wishing to be bound by theunderlying theory of the invention, it is thought that the longer dsRNAspecies serve as a substrate for the enzyme Dicer in the cytoplasm of acell. In addition to cleaving the dsRNA of the invention into shortersegments, Dicer is thought to facilitate the incorporation of asingle-stranded cleavage product derived from the cleaved dsRNA into theRISC complex that is responsible for the destruction of the cytoplasmicRNA derived from the target gene. The studies described herein haveshown that the cleavability of a dsRNA species by Dicer corresponds withincreased potency and duration of action of the dsRNA species.

In a second embodiment of the first aspect of the present invention, thedsRNA, i.e., the precursor RNAi molecule, has several properties whichenhance its processing by Dicer. According to this embodiment, the dsRNAhas a length sufficient such that it is processed by Dicer to produce anactive siRNA and at least one of the following properties: (i) the dsRNAis asymmetric, e.g., has a 3′ overhang on the antisense strand and (ii)the dsRNA has a modified 3′ end on the sense strand to directorientation of Dicer binding and processing of the dsRNA to an activesiRNA. According to this embodiment, the longest strand in the dsRNAcomprises 24-30 nucleotides. In one embodiment, the dsRNA is asymmetricsuch that the sense strand comprises 22-28 nucleotides and the antisensestrand comprises 24-30 nucleotides. Thus, the resulting dsRNA has anoverhang on the 3′ end of the antisense strand. The overhang is 1-3nucleotides, for example 2 nucleotides. The sense strand may also have a5′ phosphate.

In another embodiment, the sense strand is modified for Dicer processingby suitable modifiers located at the 3′ end of the sense strand, i.e.,the dsRNA is designed to direct orientation of Dicer binding andprocessing. Suitable modifiers include nucleotides such asdeoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and thelike and sterically hindered molecules, such as fluorescent moleculesand the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl groupfor the 2′-deoxyribofuranosyl sugar normally present in dNMPs. Othernucleotides modifiers could include 3′-deoxyadenosine (cordycepin),3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI),2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxynucleotides are used as the modifiers. When nucleotide modifiersare utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers aresubstituted for the ribonucleotides on the 3′ end of the sense strand.When sterically hindered molecules are utilized, they are attached tothe ribonucleotide at the 3′ end of the antisense strand. Thus, thelength of the strand does not change with the incorporation of themodifiers. In another embodiment, the invention contemplatessubstituting two DNA bases in the dsRNA to direct the orientation ofDicer processing of the antisense strand. In a further embodiment of thepresent invention, two terminal DNA bases are substituted for tworibonucleotides on the 3′-end of the sense strand forming a blunt end ofthe duplex on the 3′ end of the sense strand and the 5′ end of theantisense strand, and a two-nucleotide RNA overhang is located on the3′-end of the antisense strand. This is an asymmetric composition withDNA on the blunt end and RNA bases on the overhanging end.

The sense and antisense strands anneal under biological conditions, suchas the conditions found in the cytoplasm of a cell. In addition, aregion of one of the sequences, particularly of the antisense strand, ofthe dsRNA has a sequence length of at least 19 nucleotides, whereinthese nucleotides are in the 21-nucleotide region adjacent to the 3′ endof the antisense strand and are sufficiently complementary to anucleotide sequence of the RNA produced from the target gene.

Further in accordance with this embodiment, the dsRNA, i.e., theprecursor RNAi molecule, may also have one or more of the followingadditional properties: (a) the antisense strand has a right shift fromthe typical 21 mer, (b) the strands may not be completely complementary,i.e., the strands may contain simple mismatch pairings and (c) basemodifications such as locked nucleic acid(s) may be included in the 5′end of the sense strand. A “typical” 21 mer siRNA is designed usingconventional techniques. In one technique, a variety of sites arecommonly tested in parallel or pools containing several distinct siRNAduplexes specific to the same target with the hope that one of thereagents will be effective (Ji et al., 2003). Other techniques usedesign rules and algorithms to increase the likelihood of obtainingactive RNAi effector molecules (Schwarz et al., 2003; Khvorova et al.,2003; Ui-Tei et al., 2004; Reynolds et al., 2004; Krol et al., 2004;Yuan et al., 2004; Boese et al., 2005). High throughput selection ofsiRNA has also been developed (U.S. published patent application No.2005/0042641 A1, incorporated herein by reference). Potential targetsites can also be analyzed by secondary structure predictions (Heale etal., 2005). This 21 mer is then used to design a right shift to include3-9 additional nucleotides on the 5′ end of the 21 mer. The sequence ofthese additional nucleotides may have any sequence. In one embodiment,the added ribonucleotides are based on the sequence of the target gene.Even in this embodiment, full complementarity between the targetsequence and the antisense siRNA is not required.

The first and second oligonucleotides are not required to be completelycomplementary. They only need to be substantially complementary toanneal under biological conditions and to provide a substrate for Dicerthat produces a siRNA sufficiently complementary to the target sequence.Locked nucleic acids, or LNA's, are well known to a skilled artisan(Elman et al., 2005; Kurreck et al., 2002; Crinelli et al., 2002;Braasch and Corey, 2001; Bondensgaard et al., 2000; Wahlestedt et al.,2000). In one embodiment, an LNA is incorporated at the 5′ terminus ofthe sense strand. In another embodiment, an LNA is incorporated at the5′ terminus of the sense strand in duplexes designed to include a 3′overhang on the antisense strand.

In one embodiment, the dsRNA has an asymmetric structure, with the sensestrand having a 25-base pair length, and the antisense strand having a27-base pair length with a 2 base 3′-overhang. In another embodiment,this dsRNA having an asymmetric structure further contains 2deoxynucleotides at the 3′ end of the sense strand in place of two ofthe ribonucleotides.

Suitable dsRNA compositions that contain two separate oligonucleotidescan be linked by a third structure. The third structure will not blockDicer activity on the dsRNA and will not interfere with the directeddestruction of the RNA transcribed from the target gene. In oneembodiment, the third structure may be a chemical linking group. Manysuitable chemical linking groups are known in the art and can be used.Alternatively, the third structure may be an oligonucleotide that linksthe two oligonucleotides of the dsRNA is a manner such that a hairpinstructure is produced upon annealing of the two oligonucleotides makingup the dsRNA composition. The hairpin structure will not block Diceractivity on the dsRNA and will not interfere with the directeddestruction of the RNA transcribed from the target gene.

In a third embodiment of the first aspect of the present invention, thedsRNA, i.e., the precursor RNAi molecule, has several properties whichenhances its processing by Dicer. According to this embodiment, thedsRNA has a length sufficient such that it is processed by Dicer toproduce an siRNA and at least one of the following properties: (i) thedsRNA is asymmetric, e.g., has a 3′ overhang on the sense strand and(ii) the dsRNA has a modified 3′ end on the antisense strand to directorientation of Dicer binding and processing of the dsRNA to an activesiRNA. According to this embodiment, the longest strand in the dsRNAcomprises 24-30 nucleotides. In one embodiment, the sense strandcomprises 24-30 nucleotides and the antisense strand comprises 22-28nucleotides. Thus, the resulting dsRNA has an overhang on the 3′ end ofthe sense strand. The overhang is 1-3 nucleotides, such as 2nucleotides. The antisense strand may also have a 5′ phosphate.

In another embodiment, the antisense strand is modified for Dicerprocessing by suitable modifiers located at the 3′ end of the antisensestrand, i.e., the dsRNA is designed to direct orientation of Dicerbinding and processing. Suitable modifiers include nucleotides such asdeoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and thelike and sterically hindered molecules, such as fluorescent moleculesand the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl groupfor the 2′-deoxyribofuranosyl sugar normally present in dNMPs. Othernucleotide modifiers could include 3′-deoxyadenosine (cordycepin),3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI),2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxynucleotides are used as the modifiers. When nucleotide modifiersare utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers aresubstituted for the ribonucleotides on the 3′ end of the antisensestrand. When sterically hindered molecules are utilized, they areattached to the ribonucleotide at the 3′ end of the antisense strand.Thus, the length of the strand does not change with the incorporation ofthe modifiers. In another embodiment, the invention contemplatessubstituting two DNA bases in the dsRNA to direct the orientation ofDicer processing. In a further invention, two terminal DNA bases arelocated on the 3′ end of the antisense strand in place of tworibonucleotides forming a blunt end of the duplex on the 5′ end of thesense strand and the 3′ end of the antisense strand, and atwo-nucleotide RNA overhang is located on the 3′-end of the sensestrand. This is an asymmetric composition with DNA on the blunt end andRNA bases on the overhanging end.

The sense and antisense strands anneal under biological conditions, suchas the conditions found in the cytoplasm of a cell. In addition, aregion of one of the sequences, particularly of the antisense strand, ofthe dsRNA has a sequence length of at least 19 nucleotides, whereinthese nucleotides are adjacent to the 3′ end of antisense strand and aresufficiently complementary to a nucleotide sequence of the RNA producedfrom the target gene.

Further in accordance with this embodiment, the dsRNA, i.e., theprecursor RNAi molecule, may also have one or more of the followingadditional properties: (a) the antisense strand has a right shift fromthe typical 21 mer and (b) the strands may not be completelycomplementary, i.e., the strands may contain simple mismatch pairings. A“typical” 21 mer siRNA is designed using conventional techniques, suchas described above. This 21 mer is then used to design a right shift toinclude 1-7 additional nucleotides on the 5′ end of the 21 mer. Thesequence of these additional nucleotides may have any sequence. Althoughthe added ribonucleotides may be complementary to the target genesequence, full complementarity between the target sequence and theantisense siRNA is not required. That is, the resultant antisense siRNAis sufficiently complementary with the target sequence. The first andsecond oligonucleotides are not required to be completely complementary.They only need to be substantially complementary to anneal underbiological conditions and to provide a substrate for Dicer that producesa siRNA sufficiently complementary to the target sequence.

In one embodiment, the dsRNA has an asymmetric structure, with theantisense strand having a 25-base pair length, and the sense strandhaving a 27-base pair length with a 1-4 base 3′-overhang. In anotherembodiment, this dsRNA having an asymmetric structure further contains 2deoxynucleotides at the 3′ end of the antisense strand.

Suitable dsRNA compositions that contain two separate oligonucleotidescan be linked by a third structure. The third structure will not blockDicer activity on the dsRNA and will not interfere with the directeddestruction of the RNA transcribed from the target gene. In oneembodiment, the third structure may be a chemical linking group. Manysuitable chemical linking groups are known in the art and can be used.Alternatively, the third structure may be an oligonucleotide that linksthe two oligonucleotides of the dsRNA is a manner such that a hairpinstructure is produced upon annealing of the two oligonucleotides makingup the dsRNA composition. The hairpin structure will not block Diceractivity on the dsRNA and will not interfere with the directeddestruction of the RNA transcribed from the target gene.

One feature of the dsRNA compositions of the present invention is thatthey can serve as a substrate for Dicer. Typically, the dsRNAcompositions of this invention will not have been treated with Dicer,other RNases, or extracts that contain them. In the current inventionthis type of pretreatment can prevent Dicer annealing. Several methodsare known and can be used for determining whether a dsRNA compositionserves as a substrate for Dicer. For example, Dicer activity can bemeasured in vitro using the Recombinant Dicer Enzyme Kit (GTS, SanDiego, Calif.) according to the manufacturer's instructions. Diceractivity can be measured in vivo by treating cells with dsRNA andmaintaining them for 24 h before harvesting them and isolating theirRNA. RNA can be isolated using standard methods, such as with theRNeasy™ Kit (Qiagen) according to the manufacturer's instructions. Theisolated RNA can be separated on a 10% PAGE gel which is used to preparea standard RNA blot that can be probed with a suitable labeleddeoxyoligonucleotide, such as an oligonucleotide labeled with theStarfire® Oligo Labeling System (Integrated DNA Technologies, Inc.,Coralville, Iowa).

The effect that a dsRNA has on a cell can depend upon the cell itself.In some circumstances a dsRNA could induce apoptosis or gene silencingin one cell type and not another. Thus, it is possible that a dsRNAcould be suitable for use in one cell and not another. To be considered“suitable” a dsRNA composition need not be suitable under all possiblecircumstances in which it might be used, rather it need only be suitableunder a particular set of circumstances.

Modifications can be included in the dsRNA, i.e., the precursor RNAimolecule, of the present invention so long as the modification does notprevent the dsRNA composition from serving as a substrate for Dicer. Inone embodiment, one or more modifications are made that enhance Dicerprocessing of the dsRNA. In a second embodiment, one or moremodifications are made that result in more effective RNAi generation. Ina third embodiment, one or more modifications are made that support agreater RNAi effect. In a fourth embodiment, one or more modificationsare made that result in greater potency per each dsRNA molecule to bedelivered to the cell. Modifications can be incorporated in the3′-terminal region, the 5′-terminal region, in both the 3′-terminal and5′-terminal region or in some instances in various positions within thesequence. With the restrictions noted above in mind any number andcombination of modifications can be incorporated into the dsRNA. Wheremultiple modifications are present, they may be the same or different.Modifications to bases, sugar moieties, the phosphate backbone, andtheir combinations are contemplated. Either 5′-terminus can bephosphorylated.

Examples of modifications contemplated for the phosphate backboneinclude phosphonates, including methylphosphonate, phosphorothioate, andphosphotriester modifications such as alkylphosphotriesters, and thelike. Examples of modifications contemplated for the sugar moietyinclude 2′-alkyl pyrimidine, such as 2′-O-methyl, 2′-fluoro, amino, anddeoxy modifications and the like (see, e.g., Amarzguioui et al., 2003).Examples of modifications contemplated for the base groups includeabasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil,5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil and the like.Locked nucleic acids, or LNA's, could also be incorporated. Many othermodifications are known and can be used so long as the above criteriaare satisfied. Examples of modifications are also disclosed in U.S. Pat.Nos. 5,684,143, 5,858,988 and 6,291,438 and in U.S. published patentapplication No. 2004/0203145 A1, each incorporated herein by reference.Other modifications are disclosed in Herdewijn (2000), Eckstein (2000),Rusckowski et al. (2000), Stein et al. (2001); Vorobjev et al. (2001).

One or more modifications contemplated can be incorporated into eitherstrand. The placement of the modifications in the DsiRNA can greatlyaffect the characteristics of the DsiRNA, including conferring greaterpotency and stability, reducing toxicity, enhance Dicer processing, andminimizing an immune response. In one embodiment, the antisense strandor the sense strand or both strands have one or more 2′-O-methylmodified nucleotides. In another embodiment, the antisense strandcontains 2′-O-methyl modified nucleotides. In another embodiment, theantisense stand contains a 3′ overhang that is comprised of 2′-O-methylmodified nucleotides. The antisense strand could also include additional2′-O-methyl modified nucleotides.

Additionally, the dsRNA structure can be optimized to ensure that theoligonucleotide segment generated from Dicer's cleavage will be theportion of the oligonucleotide that is most effective in inhibiting geneexpression. For example, in one embodiment of the invention a 27-bpoligonucleotide of the dsRNA structure is synthesized wherein theanticipated 21 to 22-bp segment that will inhibit gene expression islocated on the 3′-end of the antisense strand. The remaining baseslocated on the 5′-end of the antisense strand will be cleaved by Dicerand will be discarded. This cleaved portion can be homologous (i.e.,based on the sequence of the target sequence) or non-homologous andadded to extend the nucleic acid strand.

In one embodiment, the sense strand comprises 24-26 nucleotides, whereinthe 2 nucleotides on the 3′ end of the sense strand aredeoxyribonucleotides. The sense strand contains a phosphate at the 5′end. The antisense strand comprises 26-29 nucleotides and contains a 3′overhang of 1-4 nucleotides. The nucleotides comprising the 3′ overhangare modified with 2′-O-methyl. The antisense strand contains alternating2′-O-methyl modified nucleotides beginning at the first monomer of theantisense strand adjacent to the 3′ overhang, and extending 15-19nucleotides from the first monomer adjacent to the 3′ overhang, wherebythe remaining 21-22 base pair antisense strand post-Dicer cleavage willcontain the modified nucleotides. For example, for a 27 nucleotideantisense strand and counting the first base at the 5′-end of theantisense strand as position number 1, 2′OMe modifications would beplaced at bases 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, and 27 In oneembodiment, the DsiRNA comprises:

5′ pXXXXXXXXXXXXXXXXXXXXXXXDD 3′ Y X X X X X X X X X X X X X X X X XXXXXXXXXpwherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl modified RNA,“Y” is an overhang domain comprised of 1-4 RNA monomers that areoptionally 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is thesense strand, and the bottom strand is the antisense strand.

RNA may be produced enzymatically or by partial/total organic synthesis,and modified ribonucleotides can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, each strand is preparedchemically. Methods of synthesizing RNA molecules are known in the art,in particular, the chemical synthesis methods as described in Verma andEckstein (1998) or as described herein.

As is known, RNAi methods are applicable to a wide variety of genes in awide variety of organisms and the disclosed compositions and methods canbe utilized in each of these contexts. Examples of genes which can betargeted by the disclosed compositions and methods include endogenousgenes which are genes that are native to the cell or to genes that arenot normally native to the cell. Without limitation these genes includeoncogenes, cytokine genes, idiotype (Id) protein genes, prion genes,genes that expresses molecules that induce angiogenesis, genes foradhesion molecules, cell surface receptors, proteins involved inmetastasis, proteases, apoptosis genes, cell cycle control genes, genesthat express EGF and the EGF receptor, multi-drug resistance genes, suchas the MDR1 gene.

More specifically, the target mRNA of the invention specifies the aminoacid sequence of a cellular protein (e.g., a nuclear, cytoplasmic,transmembrane, or membrane-associated protein). In another embodiment,the target mRNA of the invention specifies the amino acid sequence of anextracellular protein (e.g., an extracellular matrix protein or secretedprotein). As used herein, the phrase “specifies the amino acid sequence”of a protein means that the mRNA sequence is translated into the aminoacid sequence according to the rules of the genetic code. The followingclasses of proteins are listed for illustrative purposes: developmentalproteins (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt familymembers, Pax family members, Winged helix family members, Hox familymembers, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2,BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS,FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN,NRAS, PIM I, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressorproteins (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF I, NF2, RB I, TP53,and WTI); and enzymes (e.g., ACC synthases and oxidases, ACP desaturasesand hydroxylases, ADP-glucose pyrophorylases, ATPases, alcoholdehydrogenases, amylases, amyloglucosidases, catalases, cellulases,chalcone synthases, chitinases, cyclooxygenases, decarboxylases,dextriinases, DNA and RNA polymerases, galactosidases, glucanases,glucose oxidases, granule-bound starch synthases, GTPases, helicases,hernicellulases, integrases, inulinases, invertases, isomerases,kinases, lactases, lipases, lipoxygenases, lysozymes, nopalinesynthases, octopine synthases, pectinesterases, peroxidases,phosphatases, phospholipases, phosphorylases, phytases, plant growthregulator synthases, polygalacturonases, proteinases and peptidases,pullanases, recombinases, reverse transcriptases, RUBISCOs,topoisomerases, and xylanases).

In one aspect, the target mRNA molecule of the invention specifies theamino acid sequence of a protein associated with a pathologicalcondition. For example, the protein may be a pathogen-associated protein(e.g., a viral protein involved in immunosuppression of the host,replication of the pathogen, transmission of the pathogen, ormaintenance of the infection), or a host protein which facilitates entryof the pathogen into the host, drug metabolism by the pathogen or host,replication or integration of the pathogen's genome, establishment orspread of infection in the host, or assembly of the next generation ofpathogen. Pathogens include RNA viruses such as flaviviruses,picornaviruses, rhabdoviruses, filoviruses, retroviruses, includinglentiviruses, or DNA viruses such as adenoviruses, poxviruses, herpesviruses, cytomegaloviruses, hepadnaviruses or others. Additionalpathogens include bacteria, fungi, helminths, schistosomes andtrypanosomes. Other kinds of pathogens can include mammaliantransposable elements. Alternatively, the protein may be atumor-associated protein or an autoimmune disease-associated protein.

The target gene may be derived from or contained in any organism. Theorganism may be a plant, animal, protozoa, bacterium, virus or fungus.See e.g., U.S. Pat. No. 6,506,559, incorporated herein by reference.

In another aspect, the present invention provides for a pharmaceuticalcomposition comprising the dsRNA of the present invention. The dsRNAsample can be suitably formulated and introduced into the environment ofthe cell by any means that allows for a sufficient portion of the sampleto enter the cell to induce gene silencing, if it is to occur. Manyformulations for dsRNA are known in the art and can be used so long asdsRNA gains entry to the target cells so that it can act. See, e.g.,U.S. published patent application Nos. 2004/0203145 A1 and 2005/0054598A1, each incorporated herein by reference. For example, dsRNA can beformulated in buffer solutions such as phosphate buffered salinesolutions, liposomes, micellar structures, and capsids. Formulations ofdsRNA with cationic lipids can be used to facilitate transfection of thedsRNA into cells. For example, cationic lipids, such as lipofectin (U.S.Pat. No. 5,705,188, incorporated herein by reference), cationic glycerolderivatives, and polycationic molecules, such as polylysine (publishedPCT International Application WO 97/30731, incorporated herein byreference), can be used. Suitable lipids include Oligofectamine,Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals,Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be usedaccording to the manufacturer's instructions.

It can be appreciated that the method of introducing dsRNA into theenvironment of the cell will depend on the type of cell and the make upof its environment. For example, when the cells are found within aliquid, one preferable formulation is with a lipid formulation such asin lipofectamine and the dsRNA can be added directly to the liquidenvironment of the cells. Lipid formulations can also be administered toanimals such as by intravenous, intramuscular, or intraperitonealinjection, or orally or by inhalation or other methods as are known inthe art. When the formulation is suitable for administration intoanimals such as mammals and more specifically humans, the formulation isalso pharmaceutically acceptable. Pharmaceutically acceptableformulations for administering oligonucleotides are known and can beused. In some instances, it may be preferable to formulate dsRNA in abuffer or saline solution and directly inject the formulated dsRNA intocells, as in studies with oocytes. The direct injection of dsRNAduplexes may also be done. For suitable methods of introducing dsRNA seeU.S. published patent application No. 2004/0203145 A1, incorporatedherein by reference.

Suitable amounts of dsRNA must be introduced and these amounts can beempirically determined using standard methods. Typically, effectiveconcentrations of individual dsRNA species in the environment of a cellwill be about 50 nanomolar or less 10 nanomolar or less, or compositionsin which concentrations of about 1 nanomolar or less can be used. Inother embodiment, methods utilize a concentration of about 200 picomolaror less and even a concentration of about 50 picomolar or less can beused in many circumstances.

The method can be carried out by addition of the dsRNA compositions toany extracellular matrix in which cells can live provided that the dsRNAcomposition is formulated so that a sufficient amount of the dsRNA canenter the cell to exert its effect. For example, the method is amenablefor use with cells present in a liquid such as a liquid culture or cellgrowth media, in tissue explants, or in whole organisms, includinganimals, such as mammals and especially humans.

Expression of a target gene can be determined by any suitable method nowknown in the art or that is later developed. It can be appreciated thatthe method used to measure the expression of a target gene will dependupon the nature of the target gene. For example, when the target geneencodes a protein the term “expression” can refer to a protein ortranscript derived from the gene. In such instances the expression of atarget gene can be determined by measuring the amount of mRNAcorresponding to the target gene or by measuring the amount of thatprotein. Protein can be measured in protein assays such as by stainingor immunoblotting or, if the protein catalyzes a reaction that can bemeasured, by measuring reaction rates. All such methods are known in theart and can be used. Where the gene product is an RNA species expressioncan be measured by determining the amount of RNA corresponding to thegene product. Several specific methods for detecting gene expression aredescribed in Example 1. The measurements can be made on cells, cellextracts, tissues, tissue extracts or any other suitable sourcematerial.

The determination of whether the expression of a target gene has beenreduced can be by any suitable method that can reliably detect changesin gene expression. Typically, the determination is made by introducinginto the environment of a cell undigested dsRNA such that at least aportion of that dsRNA enters the cytoplasm and then measuring theexpression of the target gene. The same measurement is made on identicaluntreated cells and the results obtained from each measurement arecompared.

The dsRNA can be formulated as a pharmaceutical composition whichcomprises a pharmacologically effective amount of a dsRNA andpharmaceutically acceptable carrier. A pharmacologically ortherapeutically effective amount refers to that amount of a dsRNAeffective to produce the intended pharmacological, therapeutic orpreventive result. The phrases “pharmacologically effective amount” and“therapeutically effective amount” or simply “effective amount” refer tothat amount of an RNA effective to produce the intended pharmacological,therapeutic or preventive result. For example, if a given clinicaltreatment is considered effective when there is at least a 20% reductionin a measurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 20%reduction in that parameter.

The phrase “pharmaceutically acceptable carrier” refers to a carrier forthe administration of a therapeutic agent. Exemplary carriers includesaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract. The pharmaceutically acceptable carrier of thedisclosed dsRNA composition may be micellar structures, such as aliposomes, capsids, capsoids, polymeric nanocapsules, or polymericmicrocapsules.

Polymeric nanocapsules or microcapsules facilitate transport and releaseof the encapsulated or bound dsRNA into the cell. They include polymericand monomeric materials, especially including polybutylcyanoacrylate. Asummary of materials and fabrication methods has been published (seeKreuter, 1991). The polymeric materials which are formed from monomericand/or oligomeric precursors in the polymerization/nanoparticlegeneration step, are per se known from the prior art, as are themolecular weights and molecular weight distribution of the polymericmaterial which a person skilled in the field of manufacturingnanoparticles may suitably select in accordance with the usual skill.

Suitably formulated pharmaceutical compositions of this invention can beadministered by any means known in the art such as by parenteral routes,including intravenous, intramuscular, intraperitoneal, subcutaneous,transdermal, airway (aerosol), rectal, vaginal and topical (includingbuccal and sublingual) administration. In some embodiments, thepharmaceutical compositions are administered by intravenous orintraparenteral infusion or injection.

In general a suitable dosage unit of dsRNA will be in the range of 0.001to 0.25 milligrams per kilogram body weight of the recipient per day, orin the range of 0.01 to 20 micrograms per kilogram body weight per day,or in the range of 0.01 to 10 micrograms per kilogram body weight perday, or in the range of 0.10 to 5 micrograms per kilogram body weightper day, or in the range of 0.1 to 2.5 micrograms per kilogram bodyweight per day. Pharmaceutical composition comprising the dsRNA can beadministered once daily. However, the therapeutic agent may also bedosed in dosage units containing two, three, four, five, six or moresub-doses administered at appropriate intervals throughout the day. Inthat case, the dsRNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage unit. The dosage unitcan also be compounded for a single dose over several days, e.g., usinga conventional sustained release formulation which provides sustainedand consistent release of the dsRNA over a several day period. Sustainedrelease formulations are well known in the art. In this embodiment, thedosage unit contains a corresponding multiple of the daily dose.Regardless of the formulation, the pharmaceutical composition mustcontain dsRNA in a quantity sufficient to inhibit expression of thetarget gene in the animal or human being treated. The composition can becompounded in such a way that the sum of the multiple units of dsRNAtogether contain a sufficient dose.

Data can be obtained from cell culture assays and animal studies toformulate a suitable dosage range for humans. The dosage of compositionsof the invention lies within a range of circulating concentrations thatinclude the ED₅₀ (as determined by known methods) with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound that includes the IC₅₀ (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsof dsRNA in plasma may be measured by standard methods, for example, byhigh performance liquid chromatography.

In a further aspect, the present invention relates to a method fortreating a subject having a disease or at risk of developing a diseasecaused by the expression of a target gene. In this embodiment, the dsRNAcan act as novel therapeutic agents for controlling one or more ofcellular proliferative and/or differentiative disorders, disordersassociated with bone metabolism, immune disorders, hematopoieticdisorders, cardiovascular disorders, liver disorders, viral diseases, ormetabolic disorders. The method comprises administering a pharmaceuticalcomposition of the invention to the patient (e.g., human), such thatexpression of the target gene is silenced. Because of their highspecificity, the dsRNAs of the present invention specifically targetmRNAs of target genes of diseased cells and tissues.

In the prevention of disease, the target gene may be one which isrequired for initiation or maintenance of the disease, or which has beenidentified as being associated with a higher risk of contracting thedisease. In the treatment of disease, the dsRNA can be brought intocontact with the cells or tissue exhibiting the disease. For example,dsRNA substantially identical to all or part of a mutated geneassociated with cancer, or one expressed at high levels in tumor cells,e.g. aurora kinase, may be brought into contact with or introduced intoa cancerous cell or tumor gene.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast and liver origin. Asused herein, the terms “cancer,” “hyperproliferative,” and “neoplastic”refer to cells having the capacity for autonomous growth, i.e., anabnormal state of condition characterized by rapidly proliferating cellgrowth. These terms are meant to include all types of cancerous growthsor oncogenic processes, metastatic tissues or malignantly transformedcells, tissues, or organs, irrespective of histopathologic type or stageof invasiveness. Proliferative disorders also include hematopoieticneoplastic disorders, including diseases involvinghyperplastic/neoplatic cells of hematopoictic origin, e.g., arising frommyeloid, lymphoid or erythroid lineages, or precursor cells thereof.

The present invention can also be used to treat a variety of immunedisorders, in particular those associated with overexpression of a geneor expression of a mutant gene. Examples of hematopoietic disorders ordiseases include, without limitation, autoimmune diseases (including,for example, diabetes mellitus, arthritis (including rheumatoidarthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriaticarthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis,systemic lupus erythematosis, automimmune thyroiditis, dermatitis(including atopic dermatitis and eczematous dermatitis), psoriasis,Sjogren's Syndrome, Crohn's disease, aphthous ulcer, iritis,conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma,allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis,proctitis, drug eruptions, leprosy reversal reactions, erythema nodosumleprosum, autoimmune uveitis, allergic encephalomyclitis, acutenecrotizing hemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing, loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis,uveitis posterior, and interstitial lung fibrosis), graft-versus-hostdisease, cases of transplantation, and allergy.

In another embodiment, the invention relates to a method for treatingviral diseases, including but not limited to human papilloma virus,hepatitis C, hepatitis B, herpes simplex virus (HSV), HIV-AIDS,poliovirus, and smallpox virus. dsRNAs of the invention are prepared asdescribed herein to target expressed sequences of a virus, thusameliorating viral activity and replication. The molecules can be usedin the treatment and/or diagnosis of viral infected tissue, both animaland plant. Also, such molecules can be used in the treatment ofvirus-associated carcinoma, such as hepatocellular cancer.

The dsRNA of the present invention can also be used to inhibit theexpression of the multi-drug resistance 1 gene (“MDR1”). “Multi-drugresistance” (MDR) broadly refers to a pattern of resistance to a varietyof chemotherapeutic drugs with unrelated chemical structures anddifferent mechanisms of action. Although the etiology of MDR ismultifactorial, the overexpression of P-glycoprotein (Pgp), a membraneprotein that mediates the transport of MDR drugs, remains the mostcommon alteration underlying MDR in laboratory models (Childs and Ling,1994). Moreover, expression of Pgp has been linked to the development ofMDR in human cancer, particularly in the leukemias, lymphomas, multiplemyeloma, neuroblastoma, and soft tissue sarcoma (Fan et al.). Recentstudies showed that tumor cells expressing MDR-associated protein (MRP)(Cole et al., 1992), lung resistance protein (LRP) (Scheffer et al.,1995) and mutation of DNA topoisomerase II (Beck, 1989) also may renderMDR.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.);Ausubel et al., 1992), Current Protocols in Molecular Biology (JohnWiley & Sons, including periodic updates); Glover, 1985, DNA Cloning(IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow andLane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6thEdition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. Aguide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ.of Oregon Press, Eugene, 2000).

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1 Preparation of Double-Stranded RNA Oligonucleotides

Oligonucleotide Synthesis and Purification

RNA oligonucleotides were synthesized using solid phase phosphoramiditechemistry, deprotected and desalted on NAP-5 columns (Amersham PharmaciaBiotech, Piscataway, N.J.) using standard techniques (Damha and Olgivie,1993; Wincott et al., 1995). The oligomers were purified usingion-exchange high performance liquid chromatography (IE-HPLC) on anAmersham Source 15Q column (1.0 cm×25 cm) (Amersham Pharmacia Biotech,Piscataway, N.J.) using a 15 min step-linear gradient. The gradientvaried from 90:10 Buffers A:B to 52:48 Buffers A:B, where Buffer A was100 mM Tris pH 8.5 and Buffer B was 100 mM Tris pH 8.5, 1 M NaCl.Samples were monitored at 260 nm and peaks corresponding to thefull-length oligonucleotide species were collected, pooled, desalted onNAP-5 columns, and lyophilized.

The purity of each oligomer was determined by capillary electrophoresis(CE) on a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif.).The CE capillaries had a 100 μm inner diameter and contained ssDNA 100RGel (Beckman-Coulter). Typically, about 0.6 nmole of oligonucleotide wasinjected into a capillary, ran in an electric field of 444 V/cm anddetected by UV absorbance at 260 nm. Denaturing Tris-Borate-7 M-urearunning buffer was purchased from Beckman-Coulter. Oligoribonucleotideswere at least 90% pure as assessed by CE for use in experimentsdescribed below. Compound identity was verified by matrix-assisted laserdesorption ionization time-of-flight (MALDI-TOF) mass spectroscopy on aVoyager DE™ Biospectometry Work Station (Applied Biosystems, FosterCity, Calif.) following the manufacturer's recommended protocol.Relative molecular masses of all oligomers were within 0.2% of expectedmolecular mass.

Preparation of Duplexes

Single-stranded RNA (ssRNA) oligomers were resuspended at 100 μMconcentration in duplex buffer consisting of 100 mM potassium acetate,30 mM HEPES, pH 7.5. Complementary sense and antisense strands weremixed in equal molar amounts to yield a final solution of 50 μM duplex.Samples were heated to 95° C. for 5′ and allowed to cool to roomtemperature before use. Double-stranded RNA (dsRNA) oligomers werestored at −20° C. Single-stranded RNA oligomers were stored lyophilizedor in nuclease-free water at −80° C.

Nomenclature

For consistency, the following nomenclature has been employed throughoutthe specification and Examples. Names given to duplexes indicate thelength of the oligomers and the presence or absence of overhangs. A“21+2” duplex contains two RNA strands both of which are 21 nucleotidesin length, also termed a 21 mer siRNA duplex, and having a 2 base3′-overhang. A “21-2” design is a 21 mer siRNA duplex with a 2 base5′-overhang. A 21-0 design is a 21 mer siRNA duplex with no overhangs(blunt). A “21+2UU” is a 21 mer duplex with 2-base 3′-overhang and theterminal 2 bases at the 3′-ends are both U residues (which may result inmismatch with target sequence). A “25/27” is an asymmetric duplex havinga 25 base sense strand and a 27 base antisense strand with a 2-base3′-overhang. A “27/25” is an asymmetric duplex having a 27 base sensestrand and a 25 base antisense strand.

Example 2 Increased Potency of 25 mers

This example demonstrates that dsRNAs having strands that are 25nucleotides in length or longer have surprisingly increased potency inmammalian systems than known 21 mer to 23 mer siRNAs.

During investigations of the effects of different 5′ and 3′ endstructures of dsRNAs made through bacteriophage T7 in vitrotranscription (Kim et al., 2004), we observed that some seemed to havegreater potency than synthetic 21 mer siRNAs directed to the same targetsite, and that this property seemed to correlate with length. To furtherexplore this phenomenon, we systematically studied the silencingproperties of chemically synthesized duplex RNAs of different lengthsand designs.

Cell Culture, Transfection, and EGFP Assays

HEK 293 cells were split in 24-well plates to 60% confluency in DMEMmedium 1 d before transfection. After adding the aliquot of each RNA, 50μl of Opti Media containing the reporter vectors was added. Next, 50 μlof Opti Media containing 1.5 μl of Lipofectamine 2000 (Invitrogen) wasmixed and incubated for 15 min. The cells were then added in 0.4 ml ofDMEM medium. To normalize for transfection efficiency, each assayincluded cotransfection of the target and/or duplex RNAs with eitherfirefly luciferase or a red fluorescent protein (RFP) reporter plasmid(all other assays). For the luciferase assay, the Steady Glo Luciferaseassay kit was used according to manufacturer's instructions (Promega).For RFP cotransfection, the indicated amount of EGFP reporter plasmid(pLEGFP-C1 vector, Clontech) was transfected with 20 ng of RFP reporterplasmid (pDsRed2-C1, BD Sciences). After 24 h, RFP expression wasmonitored by fluorescence microscopy. Only experiments wheretransfection efficiency was >90% (as assessed by RFP expression) wereevaluated. EGFP expression was measured 24 h later. EGFP expression wasdetermined either from the median number of EGFP-fluorescent cellsdetermined by FACS (live cells) or by fluorometer readings (cellextracts).

For EGFP assays using NIH3T3 cells stably expressing EGFP, measurementswere determined using a VersaFluor Fluorometer (Bio-Rad) usingexcitation filter D490 and emission filter D520. Before transfections,cells were seeded to approximately 30% confluency in a 24-well plate. Onday 0, cells were transfected as described above and the medium waschanged on day 1 after transfection. The first EGFP assay was carriedout on day 3. For extract preparation 1×10⁵ cells were taken and 1×10⁴cells were further propagated for the day 6 EGFP assays. On days 6 and 9the same procedure was repeated.

Extract measurements of EGFP were obtained as follows: 1×10⁵ cells weresuspended in 300 μl of PBS and sonicated for 10 s followed by a 2-minmicrocentrifugation. The supernatants were used for fluorescencemeasurements. Percentages of EGFP expression were determined relative toextracts prepared from untreated NIH3T3 cells.

Nucleic Acid Reagents

The reporter system employed EGFP either as a transfection plasmidvector pEGFP-C1 (Clontech, Palo Alto, Calif.) or as a stabletransformant in an NIH 3T3 cell line. The coding sequence of EGFP isshown in Table 1, from Genbank accession #U55763. The ATG start codonand TAA stop codons are highlighted in bold font and sites target bysiRNA reagents in this Example and other Examples are underscored.

TABLE 1 Nucleotide Sequence of EGFPatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa (SEQ ID NO: 1)

Site-1 used for siRNA targeting in EGFP for this example was:

SITE 1: (SEQ ID NO: 2) 5′GCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGC 3′.

RNA duplexes were synthesized and prepared as described in Example 1.RNA duplexes targeting EGFP Site-1 are summarized in Table 2. Somesequences had the dinucleotide sequence “UU” placed at the 3′-end of thesense strand (Elbashir et al., 2001c; Hohjoh, 2002). Mismatches thatresulted from including 3′-terminal “UU” or where a mismatch wasintentionally positioned are highlighted in bold and underscored.

TABLE 2 Summary of Oligonucleotide Reagents, EGFP Site-1 Sequence NameSEQ ID NO. 5′ GCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGC 3′ EGFP Site-1SEQ ID NO: 2 5′ GCAAGCUGACCCUGAAGUUCA EGFPS1-21-2 SEQ ID No: 3 3′UUCGACUGGGACUUCAAGUAG SEQ ID NO: 4 5′ AAGCUGACCCUGAAGUUCAUC EGFPS1-21 +0 SEQ ID No: 5 3′ UUCGACUGGGACUUCAAGUAG SEQ ID No: 6 5′CCUGAAGUUCAUCUGCACCAC EGFPS1-21 + 2(1) SEQ ID No: 7 3′UGGGACUUCAAGUAGACGUGG SEQ ID No: 8 5′ CCCUGAAGUUCAUCUGCACCA EGFPS1-21 +2(2) SEQ ID No: 9 3′ CUGGGACUUCAAGUAGACGUG SEQ ID No: 10 5′ACCCUGAAGUUCAUCUGCACC EGFPS1-21 + 2(3) SEQ ID No: 11 3′ACUGGGACUUCAAGUAGACGU SEQ ID No: 12 5′ GACCCUGAAGUUCAUCUGCAC EGFPS1-21 +2(4) SEQ ID No: 13 3′ GACUGGGACUUCAAGUAGACG SEQ ID No: 14 5′UGACCCUGAAGUUCAUCUGCA EGFPS1-21 + 2(5) SEQ ID No: 15 3′CGACUGGGACUUCAAGUAGAC SEQ ID No: 16 5′ CUGACCCUGAAGUUCAUCUGC EGFPS1-21 +2(6) SEQ ID No: 17 3′ UCGACUGGGACUUCAAGUAGA SEQ ID No: 18 5′GCUGACCCUGAAGUUCAUCUG EGFPS1-21 + 2(7) SEQ ID No: 19 3′UUCGACUGGGACUUCAAGUAG SEQ ID No: 20 5′ GCAAGCUGACCCUGAAGUUCAU UEGFPS1-23-2UU SEQ ID No: 21 3′ UUCGACUGGGACUUCAAGUAGAC SEQ ID No: 22 5′GCUGACCCUGAAGUUCAUCUGUU EGFPS1-23 + 2UU SEQ ID No: 23 3′UUCGACUGGGACUUCAAGUAGAC SEQ ID No: 24 5′ GCAAGCUGACCCUGAAGUUCAU U UEGFPS1-24-2UU SEQ ID No: 25 3′ UUCGACUGGGACUUCAAGUAGACG SEQ ID No: 26 5′GCUGACCCUGAAGUUCAUCUGCUU EGFPS1-24 + 2UU SEQ ID No: 27 3′UUCGACUGGGACUUCAAGUAGACG SEQ ID No: 28 5′ GCAAGCUGACCCUGAAGUUCAUCU UEGFPS1-25-2UU SEQ ID No: 29 3′ UUCGACUGGGACUUCAAGUAGACGU SEQ ID No: 305′ GCUGACCCUGAAGUUCAUCUGCAUU EGFPS1-25 + 2UU SEQ ID No: 31 3′UUCGACUGGGACUUCAAGUAGACGU SEQ ID No: 32 5′ AAGCUGACCCUGAAGUUCAUCUGCACEGFPS1-26 + 0 SEQ ID No: 33 3′ UUCGACUGGGACUUCAAGUAGACGUG SEQ ID No: 345′ AAGCUGACCCUGAAGUUCAUCUGC UU EGFPS1-26 + 0UU SEQ ID No: 35 3′UUCGACUGGGACUUCAAGUAGACGUG SEQ ID No: 36 5′ GCAAGCUGACCCUGAAGUUCAUCU UUEGFPS1-26-2UU SEQ ID No: 37 3′ UUCGACUGGGACUUCAAGUAGACGUG SEQ ID No: 385′ GCUGACCCUGAAGUUCAUCUGCACUU EGFPS1-26 + 2UU SEQ ID No: 39 3′UUCGACUGGGACUUCAAGUAGACGUG SEQ ID No: 40 5′ AAGCUGACCCUGAAGUUCAUCUGCACCEGFPS1-27 + 0 SEQ ID No: 41 3′ UUCGACUGGGACUUCAAGUAGACGUGG SEQ ID No: 425′ F-AAGCUGACCCUGAAGUUCAUCUGCACC EGFPS1-27 + 0 SEQ ID No: 43 3′UUCGACUGGGACUUCAAGUAGACGUGG-F FAM #1 SEQ ID No: 44 5′AAGCUGACCCUGAAGUUCAUCUGCACC-F EGFPS1-27 + 0 SEQ ID No: 45 3′UUCGACUGGGACUUCAAGUAGACGUGG-F FAM #2 SEQ ID No: 44 5′F-AAGCUGACCCUGAAGUUCAUCUGCACC EGFPS1-27 + 0 SEQ ID No: 43 3′F-UUCGACUGGGACUUCAAGUAGACGUGG FAM #4 SEQ ID No: 46 5′AAGCUGACCCUGAAGUUCAUCUGCACC-F EGFPS1-27 + 0 SEQ ID No: 45 3′F-UUCGACUGGGACUUCAAGUAGACGUGG FAM #5 SEQ ID No: 46 5′AAGCUGACCCUGAAGUUCAUCUGCA UU EGFPS1-27 + 0UU SEQ ID No: 47 3′UUCGACUGGGACUUCAAGUAGACGUGG SEQ ID No: 48 5′ GCAAGCUGACCCUGAAGUUCAUCUGUU EGFPS1-27-2UU SEQ ID No: 49 3′ UUCGACUGGGACUUCAAGUAGACGUGGSEQ ID No: 50 5′ GCUGACCCUGAAGUUCAUCUGCAC A UU EGFPS1-27 + 2UU/SEQ ID No: 51 3′ UUCGACUGGGACUUCAAGUAGACGUGG 25 SEQ ID No: 52 5′AAGCUGACCCUGAAG A UCAUCUGCA UU EGFPS1-27 + 0UU/ SEQ ID No: 53 3′UUCGACUGGGACUUC U AGUAGACGUGG 16 SEQ ID No: 54 5′ AAGCUGACCCUGAAG AACAUCUGCA UU EGFPS1-27 + 0UU/ SEQ ID No: 55 3′ UUCGACUGGGACUUC UUGUAGACGUGG 16, 17 SEQ ID No: 56 5′ AAGCUGACCCUGAA CAA CAUCUGCA UUEGFPS1-27 + 0UU/ SEQ ID No: 57 3′ UUCGACUGGGACUU GUU GUAGACGUGG15, 16, 17 SEQ ID No: 58 5′ AAGCUGACCCUG UUCA UCAUCUGCACC EGFPS1-27 +0-mut SEQ ID No: 59 3′ UUCGACUGGGAC AAGU AGUAGACGUGG SEQ ID No: 60 5′AAGCUGACCCUGAAGUUCAUCUGCACCA EGFPS1-28 + 0 SEQ ID No: 61 3′UUCGACUGGGACUUCAAGUAGACGUGGU SEQ ID No: 62 5′AAGCUGACCCUGAAGUUCAUCUGCACCAC EGFPS1-29 + 0 SEQ ID No: 63 3′UUCGACUGGGACUUCAAGUAGACGUGGUG SEQ ID No: 64 5′AAGCUGACCCUGAAGUUCAUCUGCACCACC EGFPS1-30 + 0 SEQ ID No: 65 3′UUCGACUGGGACUUCAAGUAGACGUGGUGG SEQ ID No: 66 5′AAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAA EGFPS1-35 + 0 SEQ ID NO: 67 3′UUCGACUGGGACUUCAAGUAGACGUGGUGGCCGUU SEQ ID NO: 68 5′AAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGC EGFPS1-40 + 0 SEQ ID NO: 69 3′UUCGACUGGGACUUCAAGUAGACGUGGUGGCCGUUCGACG SEQ ID NO: 70 5′AAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUG EGFPS1-45 + 0SEQ ID NO: 71 3′ UUCGACUGGGACUUCAAGUAGACGUGGUGGCCGUUCGACGGGCACSEQ ID NO: 72

Results

An expanded set of synthetic RNA duplexes of varying length containing3′ overhangs, 5′ overhangs or blunt ends were tested for their relativepotency in a model system targeting ‘site 1’ in EGFP (Kim et al., 2003).We carried out our initial transfections using 50 nM of the various RNAduplexes (FIG. 1A). The real potency of the longer duplexes was shownonly when transfections were done at subnanomolar concentrations. Usingduplex RNA concentrations of 200 pM (FIG. 1B) and 50 pM (FIG. 1C), weobserved that potency increased with length. Blunt, 5′-overhanging and3′-overhanging ends were of similar potency. Increased potencies of thelonger duplex RNAs were observed even in the NIH3T3 cells stablyexpressing EGFP (FIG. 1F). Duplexes longer than 27 nt were synthesizedand tested for RNAi efficacy (FIG. 1D). Maximal inhibitory activity wasseen at a duplex length of 27 bp (FIG. 1D). Longer duplexes showedprogressive loss of functional RNAi activity and by 40-45 bp were whollyinactive at the tested concentrations, which also correlated with poorin vitro cleavage of these duplexes by Dicer (FIG. 1E).

Example 3 Dicer Substrates

This example demonstrates a method for determining whether a dsRNAserves as a substrate for Dicer.

In Vitro Dicer Cleavage Assays

RNA duplexes (100 pmol) were incubated in 20 μl of 20 mM Tris pH 8.0,200 mM NaCl, 2.5 mM MgCl₂ with 1 unit of recombinant human Dicer(Stratagene) for 24 h. A 3 μl aliquot of each reaction (15 pmol RNA) wasseparated in a 15% nondenaturing polyacrylamide gel, stained withGelStar (Ambrex) and visualized using UV excitation.

Results

Incubation of 21-bp to 27-bp RNA duplexes with recombinant human Dicerresulted in cleavage of the 23 mer, 25 mer and 27 mer duplexes, but notthe 21 mer duplex (FIG. 2A). Determinations of relative efficiencies ofDicer cleavage were not possible under the in vitro conditionsrecommended by the supplier owing to the slow kinetics of this reaction.Aside from the possibility that the dsRNAs longer than 30 bp may need tobe preprocessed by Drosha, a micro RNA processing enzyme, to be goodsubstrates for Dicer, we do not have an explanation for why these longerdsRNAs are both poor substrates for Dicer and poor triggers for RNAi.

Example 4 Effect of End-Modification on Dicer Activity

The effect of end-modification of dsRNA was tested using the in vitroDicer cleavage assay described in Example 3.

For the 27 mer duplexes, we tested the effects of fluoresceinend-modification on Dicer activity and the ability to trigger RNAisilencing. RNA oligonucleotides were synthesized for the EGFPS1 sitewith 6-carboxyfluorescein (6FAM) attached to the 5′ end of the sense (S)strand, 3′ end of the S-strand, 5′ end of the antisense (AS) strand and3′ end of the AS strand. Pairwise combinations were used to make duplexRNAs (FIG. 2B). Duplex 3 was the unmodified wild-type EGFPS1 27+0duplex. The structure of the 6FAM modified duplexes are shown in Table2. RNA duplexes were incubated for 24 h with recombinant human Dicer,separated by nondenaturing polyacrylamide gel electrophoresis (PAGE),stained and visualized by UV excitation (FIG. 2C). Unlike in earlierexperiments, in which RNA duplexes were fully cleaved during a 24-hincubation (FIG. 2A), all of the modified duplexes showed some degree ofresistance to cleavage by Dicer. Only the unmodified wild-type sequence(duplex 3) was fully cleaved in the in vitro Dicer reaction. The duplexbearing a 3′-6FAM on the S strand and a 3′-6FAM on the AS strand (duplex5) was totally resistant to cleavage under these conditions. Functionalpotencies of these five duplexes were compared in EGFP cotransfectionassays (FIG. 2D) using 200 pM RNA concentrations. Parallel to thepatterns seen for in vitro Dicer cleavage, all of the 27 mer duplexeswith 6FAM-modified ends were less potent than the unmodified duplexes inreducing EGFP expression. Duplexes 1, 2 and 4, which showed partialcleavage with recombinant Dicer, had three- to six-fold reduced RNAiactivity. Duplex 5, which showed no cleavage with recombinant Dicer, hadminimal RNAi activity, establishing a direct correlation between therelative effectiveness of in vitro cleavage by Dicer and RNAi in cellculture.

Example 5 In Vivo Processing by Dicer

This example confirms that the 27 mer dsRNA are processed in vivo byDicer.

Assay for Intracellular Processing of 27 Mer RNAs

RNA duplexes were transfected as described above into HEK293 cells in asix-well plate at 10 nM. After 14 h, total RNA was prepared as describedbelow. First, 20 μg of total RNA was heated for 10 min at 75° C. andmixed with ³²P 5′-end-labeled oligonucleotide probe(5′-ACCCTGAAGTTCATCTGCACC-3; SEQ ID NO:73) and hybridized in 150 mMNaCl, 50 mM Tris-HCl, pH. 7.4, 1 mM EDTA) at 24° C. Samples were loadedon 7.5% nondenaturing polyacrylamide gel and separated at 200 V for 3 hat 4° C., and the gel was exposed to X-ray film. ³²P-end-labeled 27 merand 21 mer duplex RNA oligos were used as size standards.

Results

To confirm that the 27 mer dsRNAs are processed by Dicer in vivo, wetransfected HEK293 cells with 10 nM of the duplex 3 (unmodified) orduplex 5 (both 3′ ends modified with 6FAM). After 14 h, total RNA wasisolated and hybridized with a ³²P end labeled 21 mer probe oligo (Sstrand). RNA was separated by nondenaturing PAGE and visualized byautoradiography (FIG. 2E). Similar to the results seen with in vitroDicer cleavage, in RNA prepared from cells transfected with duplex 3(unmodified 27 mer), a smaller species was observed migrating with a 21mer duplex marker, consistent with Dicer cleavage product. This 21 merspecies was not detected in RNA from cells transfected with duplex 5 (3′end-modified 27 mer).

Example 6 Potency of dsRNAs

This example examines the potency of potential cleavage products of the27 mer by Dicer.

Cleavage of a 27 mer by Dicer could result in a variety of distinct 21mers depending on where cleavage occurs; it is possible that one or amix of these possible 21 mers is significantly more potent than thespecific 21 mer that we used as our standard for comparison. To testthis possibility we synthesized seven different 21 mers that could bederived from the EGFPS1 27+0 duplex, walking in single-base steps alongthe antisense strand, using the traditional 21+2 design. These sevenduplexes were tested for RNAi activity in the HEK293 cell cotransfectionassay individually and as a pool (FIG. 3A). At concentrations of 50 or200 pM, neither the individual 21 mer duplexes nor the pooled set ofseven 21 mer duplexes showed activity comparable to the 27 mer duplex.In vitro Dicer cleavage of the 27 mers before transfection did notsignificantly enhance efficacy (FIG. 3B). As an additional control, wetransfected a mutated EGFP 27 mer duplex (Table 2), EGFPS1-27+0/mut)harboring four consecutive, centrally placed mismatched bases. Themismatches virtually eliminated any RNAi activity (FIG. 3B).

Example 7 Analysis of Dicer Cleavage Products

This example analyzed the in vitro Dicer cleavage products by massspectroscopy.

Electrospray-Ionization Liquid Chromatography Mass Spectroscopy

Electrospray-ionization liquid chromatography mass spectroscopy(ESI-LCMS) of duplex RNAs before and after treatment with Dicer weredone using an Oligo HTCS system (Novatia), which consisted ofThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processingsoftware and Paradigm MS4 HPLC (Michrom BioResources). The liquidchromatography step used before injection into the mass spectrometer(LC-MS) removes most of the cations complexed with the nucleic acids;some sodium ions can remain bound to the RNA and are visualized as minor+22 or +44 species, reflecting the net mass gain seen with substitutionof sodium for hydrogen.

Results

The species that are actually produced by incubation of the EGFPS1 27+0duplex with recombinant Dicer in vitro were identified usingelectrospray ionization mass spectrometry (ESI MS) (FIGS. 4A and 4B).Calculated masses for each possible digestion product that could resultfrom in vitro Dicer cleavage are shown in Table 3. The ESI MS analysesof the in vitro cleavage products are consistent with the known activityof this enzyme.

TABLE 3 Molecular Weights of Possible 21mer DuplexesDerived from the 27mer Duplex by Dicer Processing Sequence (SEQ ID NO:)Name Mol Wt* 5′ AAGCUGACCCUGAAGUUCAUCUGCACC (41) EGFPS1-27 + 0 8552 3′UUCGACUGGGACUUCAAGUAGACGUGG (42) 8689 5′ ACCCUGAAGUUCAUCUGCACC (11)EGFPS1-21 + 2 (3) 6672** 3′ ACUGGGACUUCAAGUAGACGU (12) 6816** 5′GACCCUGAAGUUCAUCUGCAC (13) EGFPS1-21 + 2 (4) 6712** 3′GACUGGGACUUCAAGUAGACG (14) 6855 5′ UGACCCUGAAGUUCAUCUGCA (15)EGFPS1-21 + 2 (5) 6713** 3′ CGACUGGGACUUCAAGUAGAC (16) 6815** 5′CUGACCCUGAAGUUCAUCUGC (17) EGFPS1-21 + 2 (6) 6689 3′UCGACUGGGACUUCAAGUAGA (18) 6816** 5′ GCUGACCCUGAAGUUCAUCUG (19)EGFPS1-21 + 2 (7) 6729 3′ UUCGACUGGGACUUCAAGUAG (20) 6793 *Molecularweight of 27mer is the original chemically synthesized duplex withhydroxyl ends. Calculated weights of 2lmers assume 5′phosphate on eachstrand after Dicer Processing. **Indicates masses that were consistentwith visualized peaks in FIG. 4B.

Example 8 Further Characterization of Inhibitory Properties of 27 merdsRNA

To further characterize the inhibitory properties of the 27 mer dsRNA incells stably expressing the EGFP target, stably transfected NIH3T3 cellsexpressing EGFP were transfected with 21+2 and 27+0 dsRNA duplexes (bothat 5 nM). To obtain a quantitative estimate of the duration of genesuppression, we carried out a time-course experiment, observing EGFPexpression on days 2, 4, 6, 8 and 10 after transfection. Cell extractswere prepared and measured for EGFP fluorescence using a fluorometer(FIG. 5A). EGFP suppression lasted approximately 4 d using the 21+2siRNA, consistent with previous observations (Persengiev et al., 2004),whereas inhibition obtained with the 27+0 dsRNA persisted up to 10 d. Aclass of ‘hyperfunctional’ 21+2 siRNAs has been reported showing asimilar extended duration of silencing (Reynolds et al., 2004); however,these sequences are rare and difficult to find or predict. Use of the 27mer dsRNA design may permit longer, more potent RNAi to be achieved at agreater variety of target sites.

Example 9 Effect of 27 mer dsRNA on Site Selection

A frequent problem in using RNAi as a tool to systematically inhibit theexpression of any gene is that not all target sites are equallysusceptible to suppression by siRNAs (Sherer and Rossi, 2004),necessitating complex design algorithms to predict effective sites(Reynolds et al., 2004; Ui-Tei et al., 2004; Amarzguioui and Prydz,2004). We therefore asked whether the increased potency of the 27 merdsRNA permits effective targeting at sites that are not active usingtraditional 21 mer siRNAs. Duplex RNAs were made having 21+2 and 27+0designs to two sites in EGFP (‘EGFP-S2’ and ‘EGFP-S3’) both previouslyshown to be refractory to RNAi using standard siRNAs (Kime and Rossi,2003).

Nucleic Acid Reagents

The reporter system employed EGFP as in SEQ ID NO:1 above. Site-2 (alsotermed bad site I) and Site-3 (also termed bad site 2) in EGFP weretargeted. RNA duplexes were synthesized and prepared as described inExample 1. Site-2 and Site 3 used for siRNA targeting in EGFP for thisexample were:

(SEQ ID NO: 74) SITE 2: 5′ UGAAGCAGCACGACUUCUUCAAGUCCGCCAUG 3′ and(SEQ ID NO: 75) SITE 3: 5′ UGAAGUUCGAGGGCGACACCCUGGUGAACCGCAU 3′.

RNA duplexes targeting EGFP Site-2 and EGFP Site-3 are summarized inTable 4.

TABLE 4 Summary of Oligonucleotide Reagents, EGFP Site-2 Sequence NameSEQ ID NO: 5′ UGAAGCAGCACGACUUCUUCAAGUCCGCCAUG 3′ EGFP Site-2SEQ ID NO: 74 5′ GCAGCACGACUUCUUCAAGUU EGFPS2-21 + 2 SEQ ID NO: 76 3′UUCGUCGUGCUGAAGAAGUUC SEQ ID NO: 77 5′ AAGCAGCACGACUUCUUCAAGUCCGCCEGFPS2-27 + 0 SEQ ID NO: 78 3′ UUCGUCGUGCUGAAGAAGUUCAGGCGG SEQ ID NO: 795′ UGAAGUUCGAGGGCGACACCCUGGUGAACCGCAU 3′ EGFP Site-3 SEQ ID NO: 75 5′GUUCGAGGGCGACACCCUGUU EGFPS3-21 + 2 SEQ ID NO: 80 3′UUCAAGCUCCCGCUCUGGGAC SEQ ID NO: 81 5′ GUUCGAGGGCGACACCCUGGUGAAC UUEGFPS3-27 + 0UU SEQ ID NO: 82 3′ UUCAAGCUCCCGCUCUGGGACCACUUGGCSEQ ID NO: 83

Results

The duplexes were transfected into HEK293 cells using the cotransfectionassay format (FIG. 5B) at 1 nM and 10 nM. At these doses, standard 21+2siRNAs were ineffective at both sites, whereas the 27 mer dsRNAs reducedEGFP expression by 80-90% at EGFP-S2 and by 50% (1 nM) and 80% (10 nM)at EGFP-S3. Despite the increased potency of Dicer substrate dsRNAs,empirical testing of target sites is still useful to find the mostsensitive targets for RNAi. In this regard, it is important that Dicerproducts of some 27 mers generated poorly functional siRNAs. By betterunderstanding Dicer substrate preferences, it should be possible todesign substrate RNAs that will generate the desired 21 mers. We haveobserved that a two-base 3′ overhang on only one end of the 27 mer willpreferentially guide Dicer to cleave 21-23 nt upstream of the two-base3′ overhang.

This example demonstrates that dsRNAs of the invention can efficientlytarget sites within the EGFP gene that were previously considered poortargets by previously known methods. Use of the method of the inventionwill therefore simplify site selection and design criteria for RNAi.This example also shows that the intentional placement of mismatches atthe 3′-terminus of the sense strand increases the potency of the 27 merduplex.

Example 10 Effect of 27 mer dsRNA on Other Genes

To ensure that the increased potency of the 27 mer dsRNAs was not anartifact of targeting a reporter construct, we targeted two endogenoustranscripts, human hnRNP H mRNA (Markovtsov et al., 2000) and mRNAencoding the La protein (Wolin and Cedervall, 2002).

RNAi Assays Against hnRNP H and La

HEK293 cells were plated to 30% confluency in a six-well plate. The nextday, the cells were transfected with the indicated amount of dsRNA, andthe medium was changed on the following day. The cells were harvested in300 μl PBS 72 h after transfection. Extracts were prepared as describedabove for the EGFP assays. For western blots, 2 μl of cell extract wasloaded on a 10% SDS-PAGE gel. Endogenous hnRNP H was detected using arabbit polyclonal anti-hnRNP H antibody (Markovtsov et al., 2000) andanti-rabbit antibody conjugated to alkaline phosphatase (Sigma). β-Actinwas detected with a mouse-derived anti-actin antibody (Sigma) andanti-mouse antibody conjugated to alkaline phosphatase (Sigma). Fornorthern blot analyses, harvested cells were mixed with RNA STAT-60(Tel-Test B) and total RNA was extracted according to the manufacturer'sprotocol. RNA was electrophoresed in a 6% denaturing polyacrylamide gel,transferred to a nylon membrane and probed with ³²P-end-labeled oligos(La, 5′-CCAAAGGTACCCAGCCTTCATCCAGTT-3′ (SEQ ID NO:84); β-actin,5′-GTGAGGATGCCTCTCTTGCTCTGGGCCTCG-3′ (SEQ ID NO:85)). Hybridizationswere carried out in 10 ml of hybridization solution (1 ml 50×Denhardt's,3 ml 20×SSPE, 0.5 ml 10% SDS) for 3 h at 37° C. After hybridization, theblot was washed three times with 2×SSPE at 37° C.

Nucleic Acid Reagents

The coding sequence of Homo sapiens heterogeneous nuclearribonucleoprotein H (hnRPH) mRNA (Genbank accession No. NM_(—)005520) isshown in Table 5. The ATG start codon and TAA stop codons arehighlighted in bold font and site target by siRNA reagents isunderscored.

TABLE 5 Nucleotide Sequence of HNRPHttttttttttcgtcttagccacgcagaagtcgcgtgtctagtttgtttcgacgccggaccgcgtaagagacgatgatgttgggcacggaaggtggagagggattcgtggtgaaggtccggggcttgccctggtcttgctcggccgatgaagtgcagaggtttttttctgactgcaaaattcaaaatggggctcaaggtattcgtttcatctacaccagagaaggcagaccaagtggcgaggcttttgttgaacttgaatcagaagatgaagtcaaattggccctgaaaaaagacagagaaactatgggacacagatatgttgaagtattcaagtcaaacaacgttgaaatggattgggtgttgaagcatactggtccaaatagtcctgacacggccaatgatggctttgtacggcttagaggacttccctttggatgtagcaaggaagaaattgttcagttcttctcagggttggaaatcgtgccaaatgggataacattgccggtggacttccaggggaggagtacgggggaggccttcgtgcagtttgcttcacaggaaatagctgaaaaggctctaaagaaacacaaggaaagaatagggcacaggtatattgaaatctttaagagcagtagagctgaagttagaactcattatgatccaccacgaaagcttatggccatgcagcggccaggtccttatgacagacctggggctggtagagggtataacagcattggcagaggagctggctttgagaggatgaggcgtggtgcttatggtggaggctatggaggctatgatgattacaatggctataatgatggctatggatttgggtcagatagatttggaagagacctcaattactgtttttcaggaatgtctgatcacagatacggggatggtggctctactttccagagcacaacaggacactgtgtacacatgcggggattaccttacagagctactgagaatgacatttataattttttttcaccgctcaaccctgtgagagtacacattgaaattggtcctgatggcagagtaactggtgaagcagatgtcgagttcgcaactcatgaagatgctgtggcagctatgtcaaaagacaaagcaaatatgcaacacagatatgtagaactcttcttgaattctacagcaggagcaagcggtggtgcttacgaacacagatatgtagaactcttcttgaattctacagcaggagcaagcggtggtgcttatggtagccaaatgatgggaggcatgggcttgtcaaaccagtccagctacgggggcccagccagccagcagctgagtgggggttacggaggcggctacggtggccagagcagcatgagtggatacgaccaagttttacaggaaaactccagtgattttcaatcaaacattgcataggtaaccaaggagcagtgaacagcagctactacagtagtggaagccgtgcatctatgggcgtgaacggaatgggagggttgtctagcatgtccagtatgagtggtggatggggaatgtaattgatcgatcctgatcactgactcttggtcaacctttttttttttttttttttctttaagaaaacttcagtttaacagtttctgcaatacaagcttgtgatttatgcttactctaagtggaaatcaggattgttatgaagacttaaggcccagtatttttgaatacaatactcatctaggatgtaacagtgaagctgagtaaactataactgttaaacttaagttccagcttttctcaagttagttataggatgtacttaagcagtaagcgtatttaggtaaaagcagttgaattatgttaaatgttgccctttgccacgttaaattgaacactgttttggatgcatgttgaaagacatgcttttattttttttgtaaaacaatataggagctgtgtctactattaaaagtgaaacattttggcatgtttgttaattctagtttcatttaataacctgtaaggcacgtaagtttaagctttttttttttttaagttaatgggaaaaatttgagacgcaataccaatacttaggattttggtcttggtgtttgtatgaaattctgaggccttgatttaaatctttcattgtattgtgatttccttttaggtatattgcgctaagtgaaacttgtcaaataaatcctccttttaaaaactgc (SEQ ID NO: 86)

The coding sequence of the La protein mRNA (Genbank accession No.NM_(—)005520) is shown in Table 6. The ATG start codon and TAA stopcodons are highlighted in bold font and site target by siRNA reagents isunderscored.

TABLE 6 Nucleotide Sequence of La Proteinccggcggcgctgggaggtggagtcgttgctgttgctgtttgtgagcctgtggcgcggcttctgtgggccggaaccttaaagatagccgtaatggctgaaaatggtgataatgaaaagatggctgccctggaggccaaaatctgtcatcaaattgagtattattttggcgacttcaatttgccacgggacaagtttctaaaggaacagataaaactggatgaaggctgggtacctttggagataatgataaaattcaacaggttgaaccgtctaacaacagactttaatgtaattgtggaagcattgagcaaatccaaggcagaactcatggaaatcagtgaagataaaactaaaatcagaaggtctccaagcaaacccctacctgaagtgactgatgagtataaaaatgatgtaaaaaacagatctgtttatattaaaggcttcccaactgatgcaactcttgatgacataaaagaatggttagaagataaaggtcaagtactaaatattcagatgagaagaacattgcataaagcatttaagggatcaatttttgttgtgtttgatagcattgaatctgctaagaaatttgtagagacccctggccagaagtacaaagaaacagacctgctaatacttttcaaggacgattactttgccaaaaaaaatgaagaaagaaaacaaaataaagtggaagctaaattaagagctaaacaggagcaagaagcaaaacaaaagttagaagaagatgctgaaatgaaatctctagaagaaaagattggatgcttgctgaaattttcgggtgatttagatgatcagacctgtagagaagatttacacatacttttctcaaatcatggtgaaataaaatggatagacttcgtcagaggagcaaaagaggggataattctatttaaagaaaaagccaaggaagcattgggtaaagccaaagatgcaaataatggtaacctacaattaaggaacaaagaagtgacttgggaagtactagaaggagaggtggaaaaagaagcactgaagaaaataatagaagaccaacaagaatccctaaacaaatggaagtcaaaaggtcgtagatttaaaggaaaaggaaagggtaataaagctgcccagcctgggtctggtaaaggaaaagtacagtttcagggcaagaaaacgaaatttgctagtgatgatgaacatgatgaacatgatgaaaatggtgcaactggacctgtgaaaagagcaagagaagaaacagacaaagaagaacctgcatccaaacaacagaaaacagaaaatggtgctggagaccagtagtttagtaaaccaattttttattcattttaaataggttttaaacgacttttgtttgcggggcttttaaaaggaaaaccgaattaggtccacttcaatgtccacctgtgagaaaggaaaaatttttttgttgtttaacttgtctttttgttatgcaaatgagatttctttgaatgtattgttctgtttgtgttatttcagatgattcaaatatcaaaaggaagattcttccattaaattgcctttgtaatatgagaatgtattagtacaaactaactaataaaatatatactatatgaaaagagc (SEQ ID NO: 87)

RNA duplexes were synthesized and prepared as described in Example 1.RNA duplexes targeting HNRPH1 are summarized in Table 7.

TABLE 7 Summary of Oligonucleotide Reagents Sequence Name SEQ ID NO: 5′GUUGAACUUGAAUCAGAAGAUGAAGUCAAAUUGGC 3′ HNRPH1 Site-1 SEQ ID No: 88 5′CUUGAAUCAGAAGAUGAAGUU HNRPH1-21 + 2 SEQ ID No: 89 3′UUGAACUUAGUCUUCUACUUC SEQ ID No: 90 5′ AACUUGAAUCAGAAGAUGAAGUCAAAUHNRPH1-27 + 0 SEQ ID No: 91 3′ UUGAACUUAGUCUUCUACUUCAGUUUA SEQ ID No: 925′ AUAAAACUGGAUGAAGGCUGGGUACCUUUGGAGAU 3′ La Site-1 SEQ ID NO: 93 5′CUGGAUGAAGGCUGGGUACUU La-21 + 2 SEQ ID NO: 94 3′ UUGACCUACUUCCGACCCAUGSEQ ID NO: 95 5′ AACUGGAUGAAGGCUGGGUACCUUUUU La-21 + 2 SEQ ID NO: 96 3′UUGACCUACUUCCGACCCAUGGAAACC SEQ ID NO: 97

Results

RNA duplexes were synthesized to target randomly chosen sites in thehuman hnRNP H mRNA (analyzed by western blotting) and the mRNA encodingthe La protein (analyzed by northern blotting (FIGS. 5C and 5D). Forboth targets the 27 mer duplex was more potent than the 21 mer siRNAstargeting these messages.

Example 11 Sequence Specificity of 27 mer

As a test for the sequence specificity of the 27 mer dsRNA, a series of27+0 dsRNAs with one, two or three mismatches to the EGFP target mRNAwere synthesized and tested at concentrations of 0 nM, 1 nM and 200 pMin the cotransfection assay (FIG. 6). The sequences of the mutated 27+0dsRNAs are shown in Table 2. At 200 pM, each of the mismatched sequenceswas less potent than the wild-type 27 mer dsRNA; the triple mismatch 27mer dsRNA was completely ineffective at triggering RNAi at allconcentrations tested. Similar results were obtained using a 27 merdsRNA targeted to ‘site 2’ of EGFP.

Example 12 Lack of Interferon Response

This example demonstrates that the dsRNA duplexes of the invention donot activate the interferon response.

Interferon and PKR Assays

After transfection of 293 cells with 20 nM of each RNA as describedpreviously, medium was collected after 24 h and used for ELISA assays ofinterferon α and β as previously described (Kim et al., 2004). The PKRactivation assay was done as previously described (Gunnery and Mathews,1998). PKR in the lysate was first activated by co-incubation of theindicated RNAs and radiolabeled by its auto-kinase reaction. Theradiolabeled PKR was then immunoprecipitated for analysis. To determinethe activity of PKR in the cell lysate without prior activation, dsRNAwas omitted. The reaction was incubated at 30° C. for 20 min. PKR fromthe reaction was immunoprecipitated using polyclonal antibody. Thepolyclonal antibody was added to the reaction, which was then placed onice for 1 h, followed by addition of 50 μl of 10% protein A-Sepharose inIPP500 (10 mM Tris, pH 8, 500 mM NaCl, 0.1% Nonidet P-40). This mixturewas rocked for 30 min at 4° C. The protein A-Sepharose beads were washedwith 1 ml IPP100 buffer (10 mM Tris, pH 8, 100 mM NaCl, 0.1% NonidetP-40) five times. After the last wash, the beads were boiled in proteinsample buffer and loaded onto an SDS-polyacrylamide gel followed byautoradiography.

Results

A potential problem in the use of the longer dsRNAs is activation of PKRand induction of interferons (Manche et al., 1992). We thereforeassessed whether a transfected 27 mer dsRNA activates interferon-α (FIG.7A) or interferon-β (FIG. 7B). As a positive control for interferoninduction, we transfected a triphosphate-containing single-stranded RNAwhich potently activated interferon-α and -β, as reported previously(Kim et al., 2004). Neither cytokine was detected when either the 21+2siRNA or 27+0 dsRNA was used. We have extended this observation to twoother 27 mer sequences specific for the EGFP-S2 and EGFP-S3 sites. PKRactivation in cell lysates was also assayed as described previously(Gunnery and Mathews, 1998). The lysate was treated with the indicatedRNAs, followed by immunoprecipitation. The positive control long dsRNAelicited PKR activation, but none of the shorter RNAs activated PKR(FIG. 7C).

Example 13 Asymmetric 27 mer Duplex Design and Base Modifications canInfluence Dicing Patterns and Allow Intelligent Design of 27 mer

This examples demonstrates that multiple species are produced by Diceraction on the 27 mer and that design of the 27 mer and/or inclusion ofbase modifications can be employed to direct these degradation patterns,limit heterogeneity, and predict end products.

It was demonstrated in Example 6, FIGS. 3A and 3B that all of theindividual 21 mers that could be produced by Dicer action on the EGFPS127 mer duplex are less potent in suppressing EGFP than the 27 merduplex. Nevertheless, which 21 mers are produced can influence ultimatepotency. An electrospray mass spectrometry assay was used to determinewhich 21 mers are the actual products that result from enzymaticdigestion of a 27 mer substrate RNA by Dicer. More than one 21 mer canresult from Dicer digestion of the 27 mer. Dicing patterns can becontrolled, permitting intelligent design.

Electrospray Mass Spectrometry Assay of In Vitro Dicing Reactions

RNA duplexes (100 pmoles) were incubated in 20 μl of 20 mM Tris pH 8.0,200 mM NaCl, 2.5 mM MgCl₂ with 1 unit of recombinant human Dicer(Stratagene, La Jolla, Calif.) for 12-24 hours. Electrospray-ionizationliquid chromatography mass spectroscopy (ESI-LCMS) of duplex RNAs pre-and post-treatment with Dicer were done using an Oligo HTCS system(Novatia, Princeton, N.J.), which consisted of ThermoFinnigan TSQ7000,Xcalibur data system, ProMass data processing software and Paradigm MS4™HPLC (Michrom BioResources, Auburn, Calif.). The liquid chromatographystep employed before injection into the mass spectrometer (LC-MS)removes most of the cations complexed with the nucleic acids; somesodium ion can remain bound to the RNA and are visualized as minor +22or +44 species, which is the net mass gain seen with substitution ofsodium for hydrogen. Accuracy of this assay is around +/−2 Daltons foroligonucleotides of this size.

Results

The EGFPS1-27+0 duplex was digested with Dicer and mass spectra wereobtained pre-digestion (FIG. 8A) and post-digestion (FIG. 8B). Ingeneral, Dicer will cleave a 27 mer duplex into 21 mer length fragmentswith a 5′-phosphate. Lesser amounts of 22 mers are also usuallygenerated. Small amounts of 20 mer and 23 mers can also sometimes beobserved. By comparing observed masses with the starting sequence, itcan be deduced that 4 duplexes with 2-base 3′-overhangs and 5′-phosphatewere produced in this dicing reaction. These species represent two majorcleavage products, both of which resulted in 21 mer and 22 mer duplexes.Lower case “p” represents a phosphate group. Calculated masses for eachpossible digestion product that could result from in vitro Dicercleavage are shown in Table 8.

TABLE 8 Molecular Weights of Possible DuplexesDerived from the 27mer Duplex by Dicer Processing Sequence (SEQ ID NO:)Name Mol Wt 5′ AACCUGACCCUGAAGUUCAUCUGCACC (41) EGFPS1-27 + 0 L 8552 3′UUCGACUGGGACUUCAAGUAGACGUGG (42) 8689 5′ pACCCUGAAGUUCAUCUGCACC (11)EGFPS1-21 + 2 (3) 6672 3′ ACUGGGACUUCAAGUAGACCUp (12) 6816 5′pGACCCUGAAGUUCAUCUGCACC (98) EGFPS1-22 + 2 (3) 7017 3′GACUGGGACUUCAAGUAGACCUp (99) 7161 5′ pUGACCCUGAAGUUCAUCUGCA (15)EGFPS1-21 + 2 (5) 6713 3′ CGACUGGGACUUCAAGUAGACp (16) 6815 5′pCUGACCCUGAAGUUCAUCUGCA (100) EGFPS1-22 + 2 (5) 7018 3′UCGACUGGGACUUCAAGUAGACp (101) 7121

It was demonstrated in Example 2, FIGS. 1A-1C that blunt duplexes orduplexes with 5′-overhangs can show similar or better potency thanduplexes with 3′-overhangs. In these studies, both ends of the duplexwere symmetric (i.e., both ends were blunt, both ends were 3′-overhang,or both ends were 5′-overhang). In similar studies, it was found that ablunt 27 mer duplex with two bases mismatch at one end had higherpotency than the symmetric blunt, 3′-overhang, or 5′-overhang speciestested. The blunt duplex with 2-base mismatch on one end might mimic thebehavior of an asymmetric duplex with a 2-base 3′-overhang on one endand blunt on the other end. Asymmetric 27 mer duplexes of this kind weretested and found to have increased potency and fewer diced products(less heterogeneity) and resulted in a more predictable pattern.

The double-stranded RNA binding domains of many enzymes oftenspecifically recognize RNA and not DNA or some modified RNAs. Insertionof DNA residues into the ends of blunt 27 mer duplexes was studied.Asymmetric designs with DNA residues in the 3′-end of one strandresulted in fewer diced products (less heterogeneity) and generallyproduced a predicable pattern which was opposite that of the asymmetric3′-overhang designs.

The EGFPS1-27+0 duplex was modified to have an asymmetric 3′-overhang onone side and 2 DNA bases at the 3′-end of the other side. A 5-phosphatewas placed on the recessed strand at the 3-overhang to mimic dicerprocessing. The final duplex of this design is show below aligned withthe original blunt 27 mer. Lower case “t” represents DNA dT base andlower case “p” represents a phosphate group. Calculated masses are shownin Table 9.

TABLE 9 Molecular Weights of Duplexes Sequence (SEQ ID NO:) Name Mol Wt5′ AAGCUGACCCUGAAGUUCAUCUGCACC (41) EGFPS1-27 + 0 L 8552 3′UUCGACUGGGACUUCAAGUAGACGUGG (42) 8689 5′AAGCUGACCCUGAAGUUCAUCUGCACC (41) EGFPS1-27/25 L 8552 3′ttCGACUGGGACUUCAAGUAGACGUp (102) 8075

Mass spectra were obtained pre-digestion (FIG. 8C) and post-digestion(FIG. 8D). Analysis of the dicing products made from the modifiedasymmetric duplex was much simpler than for the symmetric duplex(compare FIG. 8B with 8D). A single cleavage product was observed whichwas mostly 21 mer duplex with small amounts of 22 mer detectable. Lowercase “t” represents DNA dT base and lower case “p” represents aphosphate group. Calculated masses are shown in Table 10.

TABLE 10 Molecular Weights of Possible DuplexesDerived from the 27mer/25mer Duplex by Dicer Processing MolSequence (SEQ ID NO:) Name Wt 5′ AAGCUGACCCUGAAGUUCAUCUGCACC (41)EGFPS1- 8552 3′ ttCGACUGGGACUUCAAGUAGACCUp (102) 27/25 L 8075 5′pACCCUGAAGUUCAUCUGCACC (11) EGFPS1- 6672 3′ ACUGGGACUUCAAGUAGACCUp (12)21 + 2(3) 6816 3′ GACUGGGACUUCAAGUAGACCUp (99) 7161

Use of asymmetric design with a single 2-base 3′-overhang and selectiveincorporation of DNA residues simplifies the dicing reaction to yield asingle major cleavage product. This design additionally permitsprediction of the dicing pattern and allows for intelligent design of 27mers such that specific, desired 21 mers can be produced from dicing. Asdemonstration, a second 27 mer duplex, EGFPS1-27-R was studied whichoverlaps the EGFPS1-27-L sequence. Calculated masses are shown in Table11.

TABLE 11 Molecular Weights of Duplexes Mol Sequence (SEQ ID NO:) Name Wt5′ AAGCUGACCCUGAAGUUCAUCUGCACC (41) EGFPS1- 8552 3′UUCGACUGGGACUUCAAGUAGACGUGG (42) 27 + 0 L 8689 5′UGACCCUGAAGUUCAUCUGCACCACCG (103) EGFPS1- 8528 3′ACUGGGACUUCAAGUAGACGUGGUGGC (104) 27 + 0 R 8728

Mass spectra were obtained pre-digestion (FIG. 9A) and post-digestion(FIG. 9B). Analysis of the dicing products made from the EGFPS1-27+0 Rduplex showed a complex pattern similar to that seen with theEGFPS1-27+0 L duplex. Two major cleavage products were observed and both21 mer and 22 mer species were present. Very minor 20 mer species werealso seen. Lower case “p” represents a phosphate group. Calculatedmasses for each possible digestion product that could result from invitro Dicer cleavage are shown in Table 12.

TABLE 12 Molecular Weights of Possible DuplexesDerived from the 27mer Duplex by Dicer Processing MolSequence (SEQ ID NO:) Name Wt 5′ UGACCCUGAAGUUCAUCUGCACCACCG (103)EGFPS1- 8528 3′ ACUGGGACUUCAAGUAGACGUGGUGGC (104) 27 + 0 R 8728 5′pUGAAGUUCAUCUGCACCACCG (105) EGFPS1- 6712 21(1)R 5′pCCUGAAGUUCAUCUGCACCACC (106) EGFPS1- 6977 3′UGGGACUUCAAGUAGACGUGGUp (107) 22(3)R 7178 5′pACCCUGAAGUUCAUCUGCACCACC (108) EGFPS1- 6672 3′ACUGGGACUUCAAGUAGACGUGGUp (109) 27 + 0 R 6816 3′ACUGGGACUUCAAGUAGACGUGp (110) EGFPS1-  7161 22(5)R

The EGFPS1-27+0-R duplex was converted to a DNA-modified, asymmetric25/27 mer duplex as shown below and this duplex was studied in the invitro dicing assay. Lower case “cg” represents DNA dCdG base and lowercase “p” represents a phosphate group. Calculated masses are shown inTable 13.

TABLE 13 Molecular Weights of Duplexes Mol Sequence (SEQ ID NO:) Name Wt5′ UGACCCUGAAGUUCAUCUGCACCACCG (103) EGFPS1- 8528 3′ACUGGGACUUCAAGUAGACGUGGUGGC (104) 27 + 0 R 8728 5′pACCCUGAAGUUCAUCUGCACCACcg (111) EGFPS1- 7925 3′ACUGGGACUUCAAGUAGACGUGGUGGC (112) 25/27 R 8728

Mass spectra were obtained pre-digestion (FIG. 9C) and post-digestion(FIG. 9D). The DNA-modified asymmetric EGFPS1-25/25 R duplex showed aclean, single diced 21 mer species, as summarized in Table 14. Lowercase “p” represents a phosphate group.

TABLE 14 Molecular Weight of Possible DuplexDerived from the 25mer/27mer Duplex by Dicer Processing MolSequence (SEQ ID NO:) Name Wt 5′ pACCCUGAAGUUCAUCUGCACCACcg (111)EGFPS1- 7925 3′ ACUGGGACUUCAAGUAGACGUGGUGGC (112) 25/27 R 8728 5′pACCCUGAAGUUCAUCUGCACC (11) EGFPS1- 6672 3′ ACUGGGACUUCAAGUAGACCUp (12)21 + 2(3) 6816

If the results of FIGS. 8D and 9D are compared, it can be seen thatdigestion of the two different asymmetric duplexes EGFPS1-27/25 L andEGFPS1-25/27R result in formation of the same 21 mer species,EGFPS1-21(3). Lower case “t” or “cg” represents DNA bases and lower case“p” represents a phosphate group. Calculated masses are shown in Table15.

TABLE 15 Molecular Weights of Duplexes Mol Sequence (SEQ ID NO:) Name Wt5′ AAGCUGACCCUGAAGUUCAUCUGCACC (41) EGFPS1- 8552 3′ttCGACUGGGACUUCAAGUAGACCUp (102) 27/25 L 8075 5′pACCCUGAAGUUCAUCUGCACCACcg (111) EGFPS1- 7925 3′ACUGGGACUUCAAGUAGACGUGGUGGC (112) 25/27 R 8728 5′pACCCUGAAGUUCAUCUGCACC (11) EGFPS1- 6672 3′ ACUGGGACUUCAAGUAGACCUp (12)21 + 2(3) 6816

Therefore use of the DNA-modified asymmetric duplex design as taught bythe invention reduces complexity of the dicing reaction for 27 mer RNAspecies and permits intelligent design of 27 mers for use in RNAi suchthat it is possible to specifically target a desired location. Cleavageof the substrate 27 mer by Dicer results in a unique, predictable 21 merwherein one end of the 21 mer coincides with the 3′-overhang end of thesubstrate 27 mer.

Example 14 Asymmetric 27 mer Duplex Designs with Base Modifications canImprove Potency Over Blunt 27 mers

This examples demonstrates that the new asymmetric RNA duplexes astaught by the invention, having a 2-base 3′-overhang on one side andblunt with 3-base 3′-DNA modification on the other side, can improvepotency over blunt 27 mer duplexes.

It was demonstrated in Example 13, FIGS. 8A-8D and 9A-9D, that use ofasymmetric duplexes can direct dicing and result in a single major 21mer cleavage product. Further, the 27/25 L and 25/27 R asymmetricduplexes both result in the same 21 mer duplex after dicing. Since thesame 21 mer duplex is produced from each of the two asymmetric 27 mers,it would be anticipated by one skilled in the art that these compoundsshould functionally have similar potency. It is shown in this examplethat this is not the case and that the 25/27 R design unexpectedly hasincreased potency relative to both the 27/25 L duplex and the blunt 27+0duplex.

RNA duplexes targeting EGFPS2 were co-transfected into HEK293 cells withan EGFP expression plasmid and assayed for EGFP activity after 24 hoursincubation according to methods described above. Transfected duplexesare shown in Table 16. Lower case “p” represents a phosphate group andlower case bases “cc” and “gt” represent DNA bases while uppercase basesrepresent RNA.

TABLE 16 Transfected Duplexes Sequence Name SEQ ID NO: 5′GCAGCACGACUUCUUCAAGUU EGFPS2- SEQ ID NO: 76 3′ UUCGUCGUGCUGAAGAAGUUC21 + 2 SEQ ID NO: 77 5′ AAGCAGCACGACUUCUUCAAGUCC EGFPS2- SEQ ID NO: 78GCC 27 + 0 3′ UUCGUCGUGCUGAAGAAGUUCAGG SEQ ID NO: 79 CGG 5′CAUGAAGCAGCACGACUUCUUCAA EGFPS2- SEQ ID NO: 113 GUC 27/25 L 3′gtACUUCGUCGUGCUGAAGAAGUU SEQ ID NO: 114 Cp 5′ pGCAGCACGACUUCUUCAAGUCCGEGFPS2- SEQ ID NO: 115 cc 25/27 R 3′ UUCGUCGUGCUGAAGAAGUUCAGGSEQ ID NO: 79 CGG

The EGFPS2-21+2 and EGFPS2-27+0 RNA duplexes were employed previously inExample 9. The EGFPS2-27/25 L and EGFPS2-25/27 R duplexes are newasymmetric dsRNAs designed according to the present invention and targetthe same site, site II of EGFP. In the in vitro dicing electrospray massspectrometry assay as described in Example 13, both the EGFPS2-27/25 Land EGFPS2-25/27 R duplexes yield the same 21 mer product afterdigestion by Dicer, similar to the EGFPS2-21+2 duplex.

Transfection results are shown in FIG. 10A. As previously shown, theEGFPS2-21+2 duplexes had minimal activity in suppressing EGFP expressionwhile the 27+0 duplex showed significant inhibition. The 27/25 L duplexwas slightly less potent than the 27+0 duplex and the 25/27 R duplex wasmost potent. Based upon the teaching of prior art, this finding isunexpected, since both of the asymmetric duplexes produce the same 21mer species following dicing.

Similar transfections were done using the EGFPS1 duplexes 27/25 L and25/27 R. These duplexes produce the same 21 mer product, theEGFPS1-21(3) duplex, after dicing (Example 13). Transfected duplexes areshown in Table 17. Lower case “p” represents a phosphate group and lowercase bases “tt” represent DNA bases while uppercase bases represent RNA.

TABLE 17 Transfected Duplexes Sequence Name SEQ ID NO: 5′AAGCUGACCCUGAAGUUCAUCUGC EGFPS1- SEQ ID NO: 41 ACC 27/25 L 3′ttCGACUGGGACUUCAAGUAGACG SEQ ID NO: 102 Up 5′ pACCCUGAAGUUCAUCUGCACCACEGFPS1- SEQ ID NO: 111 cg 25/27 R 3′ ACUGGGACUUCAAGUAGACGUGGUSEQ ID NO: 112 GGC

EGFP expression after transfection is shown in FIG. 10B. As before, the25/27 R duplex was significantly more potent than the 27/25 L duplex inreducing EGFP expression. In a similar experiment in which the bluntended 27 mer was compared with 25/27 R duplex and 27/25 L duplex, it wasfound that the dsRNAs had the following potencies:

-   -   25/27 R duplex>27/25 L duplex>27 mer.

27 mer duplex RNAs can show significantly higher potency than 21 merduplexes in suppressing targeted gene expression. The blunt 27 mers canresult in a variety of 21 mer species after dicing, so preciseperformance of a blunt 27 mer duplex cannot always be predicted. Thenovel asymmetric duplexes of the present invention wherein one side ofthe duplex has a 2-base 3′-overhang and the other side is blunt and hasbase modifications, such as DNA, at the 3′-end, force dicing to occur ina predictable way so that precise 21 mers result. These asymmetricduplexes, i.e., 27/25 L and 25/27 R, are each also more potent than the21 mers. The asymmetric 25/27 R design is the most potent embodiment ofthe present invention.

FIG. 11 is an illustration comparing the embodiments of the presentinvention. The target gene sequence is illustrated by SEQ ID NO:116. The“typical” parent 21 mer used as an siRNA molecule is shown aligned withthe target gene sequence. Aligned with the target gene and parent 21 mersequences is the L 27 mer v2.1 containing a 3′ overhang on the sensestrand and two DNA bases at the 3′ end of the antisense strand. Thediced cleavage product is also shown. This alignment illustrates theleft shift in designing these precursor RNAi molecules. Also alignedwith the target gene and parent 21 mer sequences is the R 27 mer v2.1containing a 3′ overhang on the antisense strand and two DNA bases atthe 3′ end of the sense strand. The diced cleavage product is alsoshown. This alignment illustrates the right shift in designing theseprecursor RNAi molecules.

Example 15 Determination of Effective Dose

This example demonstrates a method for determining an effective dose ofthe dsRNA of the invention in a mammal A therapeutically effectiveamount of a composition containing a sequence that encodes a dsRNA,(i.e., an effective dosage), is an amount that inhibits expression ofthe product of the target gene by at least 10 percent. Higherpercentages of inhibition, e.g., 20, 50, 90 95, 99 percent or higher maybe desirable in certain circumstances. Exemplary doses include milligramor microgram amounts of the molecule per kilogram of subject or sampleweight (e.g., about 1 microgram per kilogram to about 5 milligrams perkilogram, about 100 micrograms per kilogram to about 0.5 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram). The compositions can be administered one or more times perweek for between about 1 to 10 weeks, e.g., between 2 to 8 weeks, orbetween about 3 to 7 weeks, or for about 4, 5, or 6 weeks, as deemednecessary by the attending physician. Treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments.

Appropriate doses of a particular dsRNA composition depend upon thepotency of the molecule with respect to the expression or activity to bemodulated. One or more of these molecules can be administered to ananimal, particularly a mammal, and especially humans, to modulateexpression or activity of one or more target genes. A physician may, forexample, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. Inaddition, it is understood that the specific dose level for anyparticular subject will depend upon a variety of other factors includingthe severity of the disease, previous treatment regimen, other diseasespresent, off-target effects of the active agent, age, body weight,general health, gender, and diet of the patient, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

The efficacy of treatment can be monitored by measuring the amount ofthe target gene mRNA (e.g. using real time PCR) or the amount of productencoded by the target gene such as by Western blot analysis. Inaddition, the attending physician can monitor the symptoms associatedwith the disease or disorder afflicting the patient and compare withthose symptoms recorded prior to the initiation of treatment.

It is clear from recent studies that the effects of RNAi are notentirely specific and that undesired ‘off-target’ effects can occur of amagnitude dependent on the concentration of siRNA (Persengiev et al.,2004). The new Dicer substrate dsRNA approach may facilitate use oflower concentrations of duplex RNA than are needed with traditional 21mer siRNAs. It is clear from published data that ‘off-target’ effectscan occur in certain cell lines using 21 mer siRNAs (Persengiev et al.,2004; Jackson et al., 2003), but these also can be minimized by usingreagents that have efficacy in the low to subnanomolar range (Persengievet al., 2004). To examine the potential for ‘off-target’ effects usingDicer substrate dsRNAs, we carried out microarray analyses comparing ansiRNA 21 mer with the 27 mer, each targeting EGFP site 1. NIH3T3 cellsthat stably express EGFP were transfected with concentrations of siRNAthat give effective target knockdowns (FIG. 2A, FIG. 7D). Total cellularRNAs were prepared from cells 24 and 48 h after transfection andanalyzed by hybridization to an oligonucleotide microarray as describedin FIG. 7D. Among the 16,282 mouse genes analyzed, only a small fractionshowed upregulation or downregulation more than twofold above or belowcontrol values (FIG. 7D). The 27 mer and 21 mer gave comparable resultsat their effective RNAi concentrations. There was an increase in thenumber of transcripts upregulated when the 27 mer was used at the higher25 nM concentration, but comparisons of the targets affected at 24versus 48 h and at 5 nM versus 25 nM showed no overlap. Rather thanspecific ‘off-target’ effects, these changes are more consistent withstatistical scatter among the 16,282 genes examined. The same assay wasrepeated using the EGFP-S2 27+0 duplex RNA with comparable results.

Given the increase in potency of the 27 mer dsRNAs relative to 21+2siRNAs, it is of interest that this observation has not been previouslyreported. Although others have used dsRNAs of up to 27 bp for RNAistudies in mammalian cells (Bohula et al., 2003; Caplen et al., 2001),no differences in efficacy were reported as compared with traditional21+2 duplexes. This discrepancy between previous studies and our own maysimply be due to differences in the concentration of dsRNAs tested.“Good” sites for 21 mer siRNAs can have potencies in the nanomolar range(Reynolds et al., 2004). When ‘good’ sites are tested at highconcentrations of transfected RNA, differences between 21 mer siRNAs and27 mer dsRNAs will most likely be small and easily overlooked. Markeddifferences in potency are best shown by testing at low nanomolar orpicomolar concentrations, something that is not routinely done in mostlaboratories.

Thus far, the 27 mer dsRNA design has shown increased RNAi potencyrelative to 21+2 siRNAs at every site examined. Within the set of 27mers studied here, however, a range of potencies is nevertheless seenbetween different target sites within the same gene (FIG. 3B). We haveshown that, even in the absence of fully optimized design rules, use ofDicer substrate dsRNA approach can increase RNAi potency relative totraditional 21+2 siRNAs. Additionally, the use of 27 mer dsRNAs allowstargeting of some sites within a given sequence that are refractory tosuppression with traditional 21 mer siRNAs. Use of Dicer substratedsRNAs to trigger RNAi should result in enhanced efficacy and longerduration of RNAi at lower concentrations of RNA than are required for21+2 applications. Consistent with our results linking Dicer cleavage toenhanced RNAi efficacy, it has recently been shown that chemicallysynthesized hairpin RNAs that are substrates for Dicer are more potentinducers of RNAi than conventional siRNAs and, moreover, that a two-base3′ overhang directs Dicer cleavage (Siolas et al., 2005).

Example 16 Survey of Modification Patterns

A site known to be potent for RNAi mediated suppression of the STAT1gene was chosen to survey a wide variety of modification patterns inboth 21 mer siRNAs and 27 mer DsiRNAs to test for relative potency andany toxic effects in tissue culture.

The 19-21 mer RNAs that were tested are listed in Table 18.

TABLE 18 19-21mer Duplexes with Various Modification Patterns (SEQ IDFIG. 12 Sequence NO:) Name 5′ pGCACCAGAGCCAAUGGAACUU 117 C 3′UUCGUGGUCUCGGUUACCUUGp 118 5′ pGCACCAGAGCCAAUGGAAC 119 E 3′CGUGGUCUCGGUUACCUUGp 120 5′ p G C A C C A G A G C C A A U G G A A C 121F 3′ C G U G G U C U C G G U U A C C U U Gp 122 5′ pGCACCAGAGCCAAUGGAAC123 G 3′ C G U G G U C U C G G U U A C C U U Gp 124 5′ p G C A C C A G AG C C A A U G G A A C 125 H 3′ CGUGGUCUCGGUUACCUUGp 126 5′ p G C A C C AG A G C C A A U G G A A C 127 I 3′ UUCGUGGUCUCGGUUACCUUGp 128 5′ pG C AC C A G A G C C A A U G G A A C 129 J 3′ C G U G G U C U C G G U U A C CU U G p 130 5′ pGCACCAGAGCCAAUGGAAC 131 K 3′ C G U G G U C U C G G U U AC C U U G p 132 5′ pG C A C C A G A G C C A A U G G A A C 133 L 3′CGUGGUCUCGGUUACCUUGp 134 5′ pG C A C C A G A G C C A A U G G A A C 135 M3′ UUCGUGGUCUCGGUUACCUUGp 136 5′ p G C A C C A G A G C C A A U G G A A C137 N 3′ C G U G G U C U C G G U U A C C U U G p 138 5′ pG C A C C A G AG C C A A U G G A A C 139 O 3′ C G U G G U C U C G G U U A C C U U Gp140 5′ p G C A CC AGAG CC AA U GGAA CUU 141 P 3′ UUC G U GG UCUC GG UU ACCUU G p 142 5′ p G C A CC AGAG CC AA U GGAA CUU 143 Q 3′UUCGUGGUCUCGGUUACCUUGp 144 5′ pGCACCAGAGCCAAUGGAACUU 145 R 3′ UUC G U GGUCUC GG UU A CCUU G p 146 5′ pGcAccAGAGccAAuGGAAcuU 147 S 3′UucGuGGucucGGuuAccuuGp 148 5′ pGcAccAGAGccAAuGGAAcuU 149 T 3′UUCGUGGUCUCGGUUACCUUGp 150 5′ pGcAccAGAGccAAuGGAAcuU 151 U 3′ UUC G U GGUCUC GG UU A CCUU G p 152 5′ pGCACCAGAGCCAAUGGAACUU 153 V 3′UucGuGGucucGGuuAccuuGp 154 5′ p G C A CC AGAG CC AA U GGAA CUU 155 W 3′UucGuGGucucGGuuAccuuGp 156 5′ p GcAccAGAGccAAuGGAAcuU 157 X 3′UucGuGGucucGGuuAccuuG p 158 5′ p GcAccAGAGccAAuGGAAcuU 159 Y 3′UUCGUGGUCUCGGUUACCUUGp 160 5′ p GcAccAGAGccAAuGGAAcuU 161 Z 3′ UUC G UGG UCUC GG UU A CCUU G p 162 5′ p GcAccAGAGccAAuGGAAcuU 163 AA 3′UucGuGGucucGGuuAccuuGp 164 5′ pGCACCAGAGCCAAUGGAACUU 165 BB 3′UucGuGGucucGGuuAccuuG p 166 5′ p G C A CC AGAG CC AA U GGAA CUU 167 CC3′ UucGuGGucucGGuuAccuuG p 168 5′ pGcAccAGAGccAAuGGAAcuU 169 DD 3′UucGuGGucucGGuuAccuuG p 170 RNA = AGCU 2'OMe RNA = AGCU 2'F = cu p =5'-phos

The RNA duplexes were transfected into HeLa cells in 24 well plateformat using Oligofectamine RNAs were transfected at 10 nM and 1 nMconcentrations in triplicate. At 24 hours post transfection, RNA wasprepared from cultures and cDNA was made; qRT-PCR assays were performedin triplicate and data were normalized to RPLP0 (acidic ribosomalprotein P0) mRNA levels, using a negative control siRNA as 100%. Theresults are listed in FIG. 12.

Some modification patterns worked well while others did not. Inparticular, there was a concordance with the results and the findings ofCzauderna et al. (NAR 31:2703 2003) relating to alternating 2′-O-Me RNApatterns. For this sequence/site, the fully modified duplex wasinactive, or at least had a very significant loss of potency.Transfections done at 10 nM did not show appreciable improvement inpotency for the highly modified species. None of the species showedobvious toxicity in cell culture within the 24 hours window studied.

The modification survey was next extended to the asymmetric 25/27 merDsiRNA duplex designs. Modified oligonucleotides were synthesized andannealed to make DsiRNA duplexes. The “dicing domain” was not modifiedso as to not block the ability of the endonuclease Dicer to cleave thesubstrate into the desired 21 mer final product as outlined in theschematic shown in FIG. 13. The modified 25-27 mer RNAs are listed inTable 19.

TABLE 19 25-27mer Duplexes with Various Modification Patterns (SEQ IDFIG. 14 Sequence NO:) Name 5′ pGCACCAGAGCCAAUGGAACUUGAtg 171 1 3′UUCGUGGUCUCGGUUACCUUGAACUAC 172 5′ pGCACCAGAGCCAAUGGAACUUGAtg 173 2 3′UU CGUGGUCUCGGUUACCUUGAACUAC 174 5′ pG C A C C A G A G C C A A U G G A ACUUGAtg 175 3 3′ UU C G U G G U C U C G G U U A C C U U G AACUAC 176 5′pG C A C C A G A G C C A A U G G A A CUUGAtg 177 4 3′UUCGUGGUCUCGGUUACCUUGAACUAC 178 5′ pGCACCAGAGCCAAUGGAACUUGAtg 179 5 3′UU C G U G G U C U C G G U U A C C U U G AACUAC 180 5′ pG C A C C A G AG C C A A U G G A A CUUGAtg 181 6 3′ UU C G U G G U C U C G G U U A C CU UGAACUAC 182 5′ pGCACCAGAGCCAAUGGAACUUGAtg 183 7 3′ UU C G U G G U C UC G G U U A C C U UGAACUAC 184 5′ p G C A CC AGAG CC AA U GGAA CUUGAtg185 8 3′ UUC G U GG UCUC GG UU A CCUUGAACUAC 186 5′ p G C A CC AGAG CCAA U GGAA CUUGAtg 187 9 3′ UUCGUGGUCUCGGUUACCUUGAACUAC 188 5′pGCACCAGAGCCAAUGGAACUUGAtg 189 10 3′ UUC G U GG UCUC GG UU A CCUUGAACUAC190 5′ pGcAccAGAGccAAuGGAAcuUGAtg 191 11 3′ UucGuGGucucGGuuAccuuGAACUAC192 5′ pGcAccAGAGccAAuGGAAcuUGAtg 193 12 3′ UUCGUGGUCUCGGUUACCUUGAACUAC194 5′ pGcAccAGAGccAAuGGAAcuUGAtg 195 13 3′ UUC G U GG UCUC GG UU ACCUUGAACUAC 196 5′ pGCACCAGAGCCAAUGGAACUUGAtg 197 14 3′UucGuGGucucGGuuAccuuGAACUAC 198 5′ p G C A CC AGAG CC AA U GGAA CUUGAtg199 15 3′ UucGuGGucucGGuuAccuuGAACUAC 200 5′ pGCACCAGAGCCAAUGGAACUUGAtg201 16 3′ UucGuGGucucGGuuAccuuGAACUAC 202 5′ p G C A CC AGAG CC AA UGGAA CUUGAtg 203 17 3′ UucGuGGucucGGuuAccuuGAACUAC 204 5′pGcAccAGAGccAAuGGAAcuUGAtg 205 18 3′ UucGuGGucucGGuuAccuuGAACUAC 206 RNA= AGCU 2′OMe RNA = AGCU 2′F = cu p = 5′-phos DNA = tg

As with the 19-21 mer duplexes, the 25-27 mer RNA duplexes weretransfected into HeLa cells in 24 well plate format using OligofectamineRNAs were transfected at 10 nM and 1 nM concentrations in triplicate. At24 hours post transfection, RNA was prepared from cultures and cDNA wasmade; qRT-PCR assays were performed in triplicate and data werenormalized to RPLP0 (acidic ribosomal protein P0) mRNA levels, using thenegative control siRNA as 100%. The results are listed in FIG. 14.

Results. In general, the more heavily modified duplexes showed reducedpotency, particularly those that were extensively modified with both2′-O-methyl and 2′-F bases. The less fully modified duplexes showedsimilar potency to the unmodified duplex. Patterns employing limited2′-O-methyl modification routinely were favorable.

Based upon the results outlined in Example 16, patterns were selectedthat demonstrated high initial potency to study in greater detail. Thefollowing DsiRNA, termed “ASm” in the following examples, was chosen forfurther testing:

(SEQ ID NO: 179) 5′ pGCACCAGAGCCAAUGGAACUUGAtg (SEQ ID NO: 207) 3′ UUC GU G G U C U C G G U U A C C U UGAACUACThe DsiRNA is identical to the duplex formed by SEQ ID NO: 179 and 180except the bases comprising the 3′ overhang on the antisense strand arenow modified with 2′-O-methyl. The ASm pattern has an antisense strandthat is comprised of 27 monomers, contains 2′-O-methyl modified overhangbases as well as 2′-O-methyl modified alternating bases and optionallycontains a 5′ phosphate, and a sense strand that is comprised of 25monomers wherein the two monomers on the 3′ end are DNA and contains a5′ phosphate.

Example 17 Study of 2′-O-methyl RNA Modification Patterns in EGFP

The next series of experiments were done using a dual-LucLuciferase/EGFP fusion reporter. DsiRNAs were designed which targeted anenhanced green fluorescent protein (EGFP) sequence which was embedded inthe 3′-UTR of the FLuc gene.

10 nM of each DsiRNA was co-transfected into HCT116 cells along with asingle plasmid encoding both the target (Renilla) and Normalizer(Firefly) luciferase coding regions. (psiCheck-GFP with the blocked 27mers targeting the GFP sequence in Renilla. The dual luciferase assay(Promega) was performed 24 hr post-transfection and Renilla activity(target) was normalized to Firefly luciferase. The DsiRNAs tested arelisted below in Table 20.

TABLE 20 DsiRNA modification patterns Sequence Duplex FIG. 15 NameSEQ ID NO: 5′ pACCCUGAAGUUCAUCUGCACCACcg #1 blocked SEQ ID NO: 208 3′ACU G G G A C U U C A A G U A G ACGUGGUG G C SEQ ID NO: 209 5′ pA C C CU G A A G U U C A U C U G C ACCACcg #2 blocked SEQ ID NO: 210 3′ ACU G GG A C U U C A A G U A G ACGUGGUGGC SEQ ID NO: 211 5′pACCCUGAAGUUCAUCUGCACCACcg #3 blocked SEQ ID NO: 208 3′ ACU G G G A C UU C A A G U A G A C GUGGUGGC SEQ ID NO: 211 5′pACCCUGAAGUUCAUCUGCACCACcg #4 blocked SEQ ID NO: 208 3′ ACU G G G A C UU C A A G U A G A C GUGGUG GC SEQ ID NO: 212 5′ pA C C C U G A A G U U CA U C U G C ACCACcg #5 blocked SEQ ID NO: 210 3′ACUGGGACUUCAAGUAGACGUGGUGGC SEQ ID NO: 213 5′ pACCCUGAAGUUCAUCUGCACCACcgunblocked SEQ ID NO: 208 3′ ACUGGGACUUCAAGUAGACGUGGUGGC 27merSEQ ID NO: 213 RNA = AGCU 2′OMe RNA = AGCU p = 5′-phos DNA = cg

As shown in FIG. 15, all single-strand modified variants worked well,while the double-stranded modified variant did not. The ASm pattern (#3blocked) performed well.

Example 18 Performance of ASm Designs in the Human HPRT1 Gene

ASm and related alternating 2′-O-methyl modification patterns of DsiRNAswere designed targeting the human HPRT gene (NM_(—)000194) at a knownpotent site. The DsiRNAs are listed in Table 21.

TABLE 21 DsiRNAs tested with the human HPRT gene SequenceDuplex FIG. 16 Name SEQ ID NO: 5′ pGCCAGACUUUGUUGGAUUUGAAAtt WTSEQ ID NO: 214 3′ UUCGGUCUGAAACAACCUAAACUUUAA SEQ ID NO: 215 5′ pG C C AG A C U U U G U U G G A U U UGAAAtt Sm/ASm SEQ ID NO: 216 3′ UUC G G U CU G A A A C A A C C U A AACUUUAA SEQ ID NO: 217 5′pGCCAGACUUUGUUGGAUUUGAAAtt ASm SEQ ID NO: 214 3′ UUC G G U C U G A A A CA A C C U A AACUUUAA SEQ ID NO: 217 5′ pGCCAGACUUUGUUGGAUUUGAAAtt ASm+SEQ ID NO: 214 3′ UUC G G U C U G A A A C A A C C U A AACUUU AASEQ ID NO: 218 5′ pGCCAGACUUUGUUGGAUUUGAAAtt ASm Total SEQ ID NO: 214 3′UUC G G U C U G A A A C A A C C U A A A C U U U A A SEQ ID NO: 219 5′pGCCAGACUUUGUUGGAUUUGAGCcg End-Mut SEQ ID NO: 220 3′UUCGGUCUGAAACAACCUAAACUCGGC SEQ ID NO: 221 5′ pGCCAGACUUUGUUGGAUUUGAGCcgEnd-Mut SEQ ID NO: 220 3′ UUC G G U C U G A A A C A A C C U A A A C U CG G C ASm Tot SEQ ID NO: 222 5′ pCUUCCUCUCUUUCUCUCCCUUGUga NegSEQ ID NO: 223 3′ AGGAAGGAGAGAAAGAGAGGGAACACU Control SEQ ID NO: 224 RNA= AGCU 2′OMe RNA = AGCU p = 5′-phos DNA = agct

Duplexes were transfected into HeLa cells using Oligofectamine at 10 nM,1 nM, and 0.1 nM concentration. HeLa cells were split in 24 well platesat 35% confluency and were transfected the next day with Oligofectamine(Invitrogen, Carlsbad, Calif.) using 1 μL per 65 μL OptiMEM I with RNAduplexes at the indicated concentrations. All transfections wereperformed in triplicate. RNA was harvested at 24 hours post transfectionusing SV96 Total RNA Isolation Kit (Promega, Madison, Wis.). RNA waschecked for quality using a Bioanalyzer 2100 (Agilent, Palo Alto,Calif.) and cDNA was prepared using 500 ng total RNA with SuperScript-IIReverse Transcriptase (Invitrogen, Carlsbad, Calif.) per manufacturer'sinstructions using both oligo-dT and random hexamer priming.Quantitative qRT-PCR was performed to assess relative knockdown of HPRTmRNA, using the RPLP0 gene as internal reference standard and negativecontrol DsiRNA as 100% reference levels.

As illustrated in FIG. 16, the ASm modification pattern was most similarin potency to the unmodified DsiRNA duplex at the same site. The “ASmTotal” duplex, which has alternating 2′OMe modifications throughout theentire AS strand, was also effective at reducing HPRT mRNA levels,albeit with lower potency than the WT or ASm versions at low dose (0.1nM). Data from in vitro dicing assays indicates that the 2′-O-methylmodification pattern employed in the “ASm Total” duplex prevents normalprocessing by Dicer (see Example 20). The most straightforwardinterpretation of these data are that the intact 27 nt AS strand of thisDsiRNA can directly load into RISC and trigger RNAi in the absence ofDicer cleavage into a 21 mer siRNA.

To test this hypothesis, the “end-mut” mutant version of the HPRT DsiRNAwas designed, wherein the terminal 4 bases of the sequence (5′-end ofthe AS strand) were mutated (see Table 21 sequence listing). This willblock hybridization of the “seed region” if the 27 mer loads intact, andshould thus block or significantly reduce activity of this duplex totrigger RNAi-mediated suppression of HPRT mRNA. If Dicer cleavage occursprior to RISC loading, the 4-base mutated domain will be cleaved off andthe resulting 21 mer product will be identical to the normal WTsequence.

The unmodified end-mut sequence showed potency similar to the WT HPRTDsiRNA, which is the expected result if Dicer cleavage occurred. The“ASm-total” version of the end-mut sequence, however, showed markedlyreduced potency, which is the expected result if the mutant sequencewere loaded into RISC without Dicer cleavage. It therefore appears thatDicer processing proceeds if the substrate dsRNA is cleavable (i.e.,does not have nuclease resistant modifications spanning the cleavagesite). If the DsiRNA is modified within the dicing site and cannot beprocessed by Dicer, the dsRNA can still function as a trigger for RNAi,presumably by RISC loading as an intact 27 mer, but functions withreduced potency. It is therefore desirable, but not required, thatmodification patterns employed for DsiRNA employ cleavable bases andinternucleoside linkages at the expected site of Dicer processing.

Example 19 Dicer Processing of ASm Modified HPRT DsiRNAs

In vitro dicing assays were performed on unmodified and modifiedduplexes. Duplexes were incubated with recombinant human Dicer andreaction products were subjected to ESI-MS (electrospray massspectrometry). RNA duplexes (100 pmol) were incubated in 20 μL of 20 mMTris pH 8.0, 200 mM NaCl, 2.5 mM MgCl₂ with or without 1 unit ofrecombinant human Dicer (Stratagene, La Jolla, Calif.) at 37° C. for 24h. Samples were desalted using a Performa SR 96 well plate (EdgeBiosystems, Gaithersburg, Md.). Electrospray-ionization liquidchromatography mass spectroscopy (ESI-LCMS) of duplex RNAs pre- andpost-treatment with Dicer were done using an Oligo HTCS system (Novatia,Princeton, N.J.), which consisted of ThermoFinnigan TSQ7000, Xcaliburdata system, ProMass data processing software and Paradigm MS4™ HPLC(Michrom BioResources, Auburn, Calif.). The liquid chromatography stepemployed before injection into the mass spectrometer (LC-MS) removesmost of the cations complexed with the nucleic acids; some sodium ioncan remain bound to the RNA and are visualized as minor +22 or +44species, which is the net mass gain seen with substitution of sodium forhydrogen. All dicing experiments were performed at least twice. Massdata are summarized in Table 22.

TABLE 22 Resulting DsiRNAspost-Dicer processing Mass SEQ ID SequenceSample (Da) NO: 5′ pGCCAGACUUUGUUGGAUUUGAAAtt WT 8037 214 3′UUCGGUCUGAAACAACCUAAACUUUAA 8546 215 5′ pGCCAGACUUUGUUGGAUUUGA WT 6771225 3′ UUCGGUCUGAAACAACCUAAAp Diced 6744 226 5′pGCCAGACUUUGUUGGAUUUGAAAtt ASm 8037 214 3′ UUC G G U C U G A A A C A A CC U A AACUUUAA 8700 217 5′ pGCCAGACUUUGUUGGAUUUGA ASm (21) 6771 227 3′UUC G G U C U G A A A C A A C C U A AAp Diced 6884 228 5′pGCCAGACUUUGUUGGAUUUGAA ASm (22) 7100 229 3′ UUC G G U C U G A A A C A AC C U A AACp Diced 7189 230 5′ pGCCAGACUUUGUUGGAUUUGAAAtt ASm Total 8037214 3′ UUC G G U C U G A A A C A A C C U A A A C U U U A A 8756 219 5′pGCCAGACUUUGUUGGAUUUGAA ASm Total 7100 231 3′ UUC G G U C U G A A A C AA C C U A A A C U U U A A Diced 8756 219 RNA = AGCU 2′OMe RNA = AGCU p =5′-phos DNA = agct

The unmodified HPRT DsiRNA diced into the expected 21 mer species. TheASm modified HPRT DsiRNA diced into an equal mix of 21 mer and 22 merproducts with cleavage occurring at the expected position. The ASm-totalmodified HPRT DsiRNA did not undergo normal Dicer processing. Theunmodified S-strand was cleaved into a 22 mer, however the 2′-O-Memodified AS strand remained uncut. The alternating 2′-O-methyl RNApattern is therefore resistant to Dicer endonuclease cleavage.

Example 20 Measuring Interferon Responses Through qRT-PCR Assays

A panel of qRT-PCR assays were developed that are specific to human andmouse genes that involve recognition of nucleic acids by the innateimmune system. Monitoring the relative levels of these genes is oneapproach to examine activation of immune pathways. The immune receptors,signaling molecules, interferon response genes (IRGs), and controls thatwere tested are listed in Table 23.

TABLE 23 Genes tested for IFN activation GENE ASSAY GENE PROPERTIESRPLP0 ribosomal protein, housekeeping gene HPRT DsiRNA target,housekeeping gene STAT1 IRG OAS1 Receptor, cytoplasmic, detects longdsRNAs IFITM1 IRG IFIT1 (p56) IRG RIG-I Receptor, cytoplasmic, detectstriphosphate and blunt RNAs MDA5 Receptor, cytoplasmic, detects shortdsRNAs TLR3 Receptor, endocytic, detects dsRNAs TLR4 Receptor, cellsurface, detects LPS TLR7 Receptor, endocytic, detects ssRNAs TLR8Receptor, endocytic, detects ssRNAs TLR9 Receptor, endocytic, detectsunmethylated CpG motif DNAs

T98G cells were studied as these cells are known to strongly respond todsRNAs in tissue culture. In particular, these cells have been shown topossess receptor/signaling pathways that can recognize longer RNAs ofthe type of the invention (Marques, et al. 2006). Two versions of ananti-HPRT DsiRNA, unmodified and ASm 2′-O-methyl modified (see Table 21of Example 18), were transfected at high dose (100 nM) using siLentFectcationic lipid (Bio-Rad Laboratories). IFN pathway genes were assayed incell culture at T=24 h post transfection using lipid alone as zerobaseline. Two versions of an anti-HPRT DsiRNA, unmodified and ASm2′-O-methyl modified (see Table 21 of Example 18), were transfected athigh dose (100 nM) using siLentFect cationic lipid (Bio-RadLaboratories). IFN pathway genes were assayed in cell culture at T=24 hpost transfection using lipid alone as zero baseline.

Real-time PCR reactions were done using an estimated 33 ng cDNA per 25μL reaction using Immolase DNA Polymerase (Bioline, Randolph, Mass.) and200 nM primers and probe. Cycling conditions employed were: 50° C. for 2minutes and 95° C. for 10 minutes followed by 40 cycles of 2-step PCRwith 95° C. for 15 seconds and 60° C. for 1 minute. PCR and fluorescencemeasurements were done using an ABI Prism™ 7000 Sequence Detector or anABI Prism™ 7900 Sequence Detector (Applied Biosystems Inc., Foster City,Calif.). All data points were performed in triplicate. Expression datawas normalized to internal control human acidic ribosomal phosphoproteinP0 (RPLP0) (NM_(—)001002) levels which were measured in separate wellsin parallel.

The results are illustrated in FIG. 17A. Treatment of T98G cells withthe unmodified DsiRNA resulted in potent stimulation of a number of IFNresponse genes and receptors within the innate immune system. Use of ASm2′-O-methyl modified RNA resulted in minimal pathway activation. FIGS.17B and 17C more clearly illustrate the control sequences. Both theRPLP0 (control) and HPRT (target) mRNAs are reduced to very low levelsin cells treated with the unmodified DsiRNA, which is evidence thatgross degradation of mRNA has taken place (typical of a Type-I IFNresponse), whereas in the ASm treated cells the control mRNA RPLP0levels are normal while the target HPRT levels is reduced (indicative ofsuccessful RNAi suppression).

Example 21 Measuring Immune Response to DsiRNAs Using Cytokine Assays

It is well known in the art that dsRNAs (including 21 mer siRNAs) are atrisk of triggering an innate immune response (classically a Type-I IFNresponse) when administered in vivo, particularly when given complexedwith lipid-based delivery reagents that maximize exposure to theendosomal compartments where Toll-Like Receptors (TLRs) 3, 7, and 8reside. Modification with 2′-O-methyl RNA in 21 mers has been shown togenerally prevent IFN activation (Judge et al., Molecular Therapy 2006).

Cytokine assays were performed using reagents from Upstate (Millipore).These are antibody-based assays which run in multiplex using the Luminexfluorescent microbead platform. Standard curves were established topermit absolute quantification. Assays were performed according to themanufacturer's recommendation.

T98G cells were studied for cytokine release following transfection withmodified and unmodified HPRT DsiRNAs. Supernatants from the samecultures that were studied for gene expression in Example 20 wereemployed, so cytokine secretion and gene expression can be directlycontrasted within the same experiment. RNAs were transfected at highdose (100 nM) using siLentFect (cationic lipid). Cytokine assays wereperformed on culture supernatants at T=24 h post transfection usinglipid alone as zero baseline.

FIG. 18 demonstrates that when using unmodified RNA, high release levelsof the inflammatory cytokines IL-8 and IL-6 were observed. When the samesequence was transfected modified with 2′-O-methyl RNA in the ASmpattern, no significant elevation of cytokine levels was observed.

Example 22 Serum Stability for DsiRNAs

The following example demonstrates the relative level of protection fromnuclease degradation that is conferred on DsiRNAs with and withoutmodifications.

RNAs were incubated in 50% fetal bovine serum for various periods oftime (up to 24 h) at 37° C. Serum was extracted and the nucleic acidswere separated on a 20% non-denaturing PAGE and visualized with Gelstarstain. Markers are blunt, duplex RNAs of the indicated lengths.Sequences studied are listed in Table 24.

TABLE 24 DsiRNA tested for stability in serum SequenceDuplex FIG. 19 Name SEQ ID NO: 5′ pGCCAGACUUUGUUGGAUUUGAAAtt WT27SEQ ID NO: 214 3′ UUCGGUCUGAAACAACCUAAACUUUAA SEQ ID NO: 215 5′pGCCAGACUUUGUUGGAUUUGAAAtt WT27 ASm SEQ ID NO: 214 3′ UUC G G U C U G AA A C A A C C U A AACUUUAA SEQ ID NO: 217 5′ GCCAGACUUUGUUGGAUUUGAHPRT 21 SEQ ID NO: 232 3′ UUCGGUCUGAAACAACCUAAA SEQ ID NO: 233 5′pGCCAGACUUUGUUGGAUUUGAAAtt WT27 ASm SEQ ID NO: 214 3′ UUC G G U C U G AA A C A A C C U A AACUUUAAp 5′P SEQ ID NO: 234 RNA = AGCU 2′OMe RNA =AGCU p = 5′-phos DNA = tt

As illustrated in FIG. 19, the 27 mer DsiRNAs showed improved stabilityin serum even without chemical modification. Addition of 2′-O-methyl RNAin the ASm pattern did not appear to improve stability by this assay.The ASm+5′P modification pattern did improve stability. The 21 mersiRNAs showed rapid degradation to species that likely represents ablunt 19 mer (first step of degradation is removal of the ssRNA3′-overhangs). Degradation of the duplex dsRNA domain is slower.

RNAs that have been subjected to serum degradation were separated byHPLC and the peaks purified; actual degradation products were identifiedby LC-MS using the Novatia ESI-MS platform (methods as describedpreviously for in vitro dicing assays). Interestingly, the band thatappears to be intact by PAGE methods (FIG. 21) actually is a mixedpopulation including intact and partially degraded RNAs. Mass analysisrevealed the presence of a 5′-exonuclease activity that is blocked by5′-phosphate, so that the duplex bearing 5′-phosphate groups on bothstrands (ASm+5′P) was more intact than the unmodified or ASm modifiedduplexes. The relative amount of intact DsiRNA present after 24 hincubation in serum was ASm+5′P>ASm>WT unmodified. 5′-phosphate ispresent on naturally occurring miRNAs and siRNAs that result from Dicerprocessing. Addition of 5′-phosphate to all strands in synthetic RNAduplexes may be an inexpensive and physiological method to confer somelimited degree of nuclease stability.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

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What is claimed is:
 1. A formulation comprising an isolated doublestranded nucleic acid capable of reducing the expression of a targetgene comprising: a first oligonucleotide strand that is 25-30nucleotides in length and contains 1-3 DNA bases on the 3′ end of thefirst oligonucleotide strand; and a second oligonucleotide strand thatis 25-30 nucleotides in length comprising an overhang of unmodifiedand/or modified nucleotides and a domain that hybridizes to the firstoligonucleotide strand comprising unmodified and modified nucleotides,wherein said second oligonucleotide strand is sufficiently complementaryto a target mRNA along at least 19 nucleotides of said secondoligonucleotide strand length to reduce target gene expression when saiddouble stranded nucleic acid is introduced into a mammalian cell,wherein said double stranded nucleic acid is present in said formulationin an amount effective to reduce target gene expression when said doublestranded nucleic acid is introduced into a mammal or a mammalian cell.2. An isolated double stranded nucleic acid comprising: a firstoligonucleotide strand that is 25-30 nucleotides in length and contains1-3 DNA bases on the 3′ end of the first oligonucleotide strand; and asecond oligonucleotide strand that is 25-30 nucleotides in lengthcomprising an overhang of unmodified and/or modified nucleotides and adomain that hybridizes to the first oligonucleotide strand, comprisingunmodified and modified nucleotides, wherein said second oligonucleotidestrand is sufficiently complementary to a target mRNA along at least 19nucleotides of said second oligonucleotide strand length to reducetarget gene expression when said double stranded nucleic acid isintroduced into a mammal or a mammalian cell.
 3. A method for reducingexpression of a target gene in a mammal or a mammalian cell comprising:administering the isolated double stranded nucleic acid of claim 2 tothe mammal or the mammalian cell in an amount sufficient to reduceexpression of a target gene.
 4. The method of claim 3, wherein thedouble stranded nucleic acid is administered to the mammalian cell invitro.
 5. An isolated double stranded nucleic acid comprising first andsecond oligonucleotide strands, said first oligonucleotide strand being25 to 30 nucleotides in length and comprising a 5′ terminus having a 5′terminal nucleotide and a 3′ terminus having a 3′ terminal nucleotide,referring to the 5′ terminal nucleotide of said first oligonucleotidestrand as position 1 and successively numbered nucleotides in thedirection 5′ to 3′ of said first oligonucleotide strand at eachconsecutive residue, wherein said second oligonucleotide strandcomprises a 5′ terminus having a 5′ terminal nucleotide and a 3′terminus having a 3′ terminal nucleotide and said second oligonucleotidestrand is 1-4 residues longer at the 3′ terminus of said secondoligonucleotide strand than the 5′ terminus of said firstoligonucleotide strand, wherein said 3′ terminal nucleotide of saidfirst oligonucleotide strand and said 5′ terminal nucleotide of saidsecond oligonucleotide strand are paired to form a paired blunt end,wherein said 5′ terminal nucleotide of said first oligonucleotide strandis paired with said second oligonucleotide strand to form a doublestranded nucleic acid comprising a single stranded overhang 1-4nucleotides in length at said 3′ terminus of said second oligonucleotidestrand such that said double stranded nucleic acid also comprises aduplex region of at least 25 nucleotide residues, wherein said secondoligonucleotide strand comprises modified nucleotide residues at eachnucleotide of said second oligonucleotide strand that is paired withsaid first oligonucleotide positions 1, 3, 5, 7, 9, 11, 13 and 15, andwherein said second oligonucleotide strand comprises a sequencecomplementary to a target mRNA and said isolated double stranded nucleicacid reduces target gene expression when introduced into a mammaliancell.
 6. The isolated double stranded nucleic acid of claim 5, furthercomprising a modified nucleotide residue at said nucleotide residue ofsaid second oligonucleotide strand that is paired with position 24 ofsaid first oligonucleotide.
 7. The isolated double stranded nucleic acidof claim 5, wherein each nucleotide of said second oligonucleotidestrand that is paired with said first oligonucleotide strand positions2, 4, 6, 8, 10, 12, 14, 16, 17, 18, 19, 20, 21, 22, 23 and 25 of saidsecond oligonucleotide is an unmodified ribonucleotide.
 8. The isolateddouble stranded nucleic acid of claim 5, wherein said secondoligonucleotide strand comprises a sequence complementary to a targetRNA along at least 19 nucleotides of said second oligonucleotide strandlength.
 9. The isolated double stranded nucleic acid of claim 5, whereinsaid 3′ terminus of said first oligonucleotide strand comprises 1-3consecutive deoxyribonucleotides starting at said 3′ terminalnucleotide.
 10. The isolated double stranded nucleic acid of claim 5,wherein said first oligonucleotide strand comprises two deoxynucleotideresidues as the ultimate and penultimate nucleotides at said 3′terminus.
 11. The isolated double stranded nucleic acid of claim 5,wherein said first oligonucleotide strand has a length which is at least26 nucleotides.
 12. The isolated double stranded nucleic acid of claim5, wherein said modified nucleotide residues of said secondoligonucleotide strand are selected from the group consisting of2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge,4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and 2′-O—(N-methylcarbamate).13. The isolated double stranded nucleic acid of claim 5, wherein eachmodified nucleotide of said second oligonucleotide strand is a2′-O-methyl ribonucleotide.
 14. The isolated double stranded nucleicacid of claim 5, wherein said 3′ overhang of said second oligonucleotidestrand comprises a modified nucleotide.
 15. The isolated double strandednucleic acid of claim 14, wherein each nucleotide of said 3′ overhang isa modified nucleotide.
 16. The isolated double stranded nucleic acid ofclaim 5, wherein said 3′ overhang is two nucleotides in length.
 17. Theisolated double stranded nucleic acid of claim 5, wherein the relativelength in nucleotide residues of said first and second strands isselected from the group consisting of: second strand 26-29 nucleotideresidues in length and first strand 25 nucleotide residues in length,second strand 27-30 nucleotide residues in length and first strand 26nucleotide residues in length, second strand 28-30 nucleotide residuesin length and first strand 27 nucleotide residues in length, secondstrand 29-30 nucleotide residues in length and first strand 28nucleotide residues in length, and second strand 30 nucleotide residuesin length and first strand 29 nucleotide residues in length.
 18. Theisolated double stranded nucleic acid of claim 5, wherein said doublestranded nucleic acid is a double stranded RNA and wherein said doublestranded RNA is cleaved endogenously in a mammalian cell by Dicer. 19.The isolated double stranded nucleic acid of claim 2, wherein themodified nucleotide comprises a modified base and/or a modified sugarmoiety.
 20. The isolated double stranded nucleic acid of claim 2,wherein the modified nucleotide is selected from the group consistingof: dideoxyribonucleotides, acyclonucleotides 3′-deoxyadenosine(cordycepin), 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine(ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T).
 21. The isolated doublestranded nucleic acid of claim 19, wherein the modified sugar moiety isselected from the group consisting of 2′-O-methyl, 2′-methoxyethoxy,2′-fluoro, 2′-allyl, 2′-O[2-(methylamino)-2-oxoethyl], 4′-thio,4′-CH2-O-2′-bridge, 4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and2′-O—(N-methylcarbamate).
 22. The isolated double stranded nucleic acidof claim 2, wherein at least one internucleoside linkage is modified.23. The isolated double stranded nucleic acid of claim 22, wherein theinternucleoside linkage modification is selected from the groupconsisting of: methylphosphonate, phosphorothioate, and phosphotriestermodifications.
 24. The isolated double stranded nucleic acid of claim 2,further comprising a fluorescein.
 25. The isolated double strandednucleic acid of claim 2, wherein the modified nucleotide comprises anLNA modification.